Pharmaceutical compositions comprising bacterial delivery vehicles and uses thereof

ABSTRACT

The present invention relates to the delivery of a payload by bacterial delivery vehicle, i.e. the encapsulation and the delivery of a single plasmid by different bacterial virus particles. More specifically, the present invention concerns a pharmaceutical composition comprising a payload packaged in at least two different bacterial delivery vehicles and a method of production thereof.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“2643-10_ST25.txt” created on Sep. 9, 2019 and is 4,739 bytes in size.The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology and particularlyto the delivery of a payload by bacterial delivery vehicle. Morespecifically, the present invention concerns the encapsulation and thedelivery of a plasmid by bacterial virus particles.

BACKGROUND OF THE INVENTION

Nowadays, the treatment of bacterial infections mainly relies on the useof antibiotics. However, excessive and inappropriate use of antibioticshas fostered the emergence and spread of antibiotic-resistantmicroorganisms. Indeed, infections caused by antibiotic-resistantmicroorganisms, also known as “superbugs”, sometimes no longer respondto conventional treatments, thereby extending the duration of thedisease related to infection and even leading to patient death. Becauseof the development of this antibiotic resistance phenomenon and the lackof discovery of new antibiotic classes, humanity is now facing thepossibility of a future without effective treatment for bacterialinfections.

Bacterial viruses (or phages) are small viruses displaying the abilityto infect and kill bacteria while they do not affect cells from otherorganisms. Initially described almost a century ago by William Twort,and independently discovered shortly thereafter by Felix d'Herelle, morethan 6000 different bacterial viruses have been discovered so far anddescribed morphologically. The vast majority of these viruses are tailedwhile a small proportion are polyhedral, filamentous or pleomorphic.They may be classified according to their morphology, their geneticcontent (DNA vs. RNA), their specific host, the place where they live(marine virus vs. other habitats), and their life cycle. Asintracellular parasites of bacterial cells, phages display differentlife cycles within the bacterial host: lytic, lysogenic,pseudo-lysogenic, and chronic infection. Lytic phages, once their DNAinjected into their host, replicate their own genome and produce newviral particles at the expense of the host. Indeed, they cause lysis ofthe host bacterial cell as a normal part of the final stage of theirlife cycles to liberate viral particles. Temperate phages (also termedtemperate phages) can either replicate by means of the lytic life cycleand cause lysis of the host bacterium, or they can incorporate their DNAinto the host bacterial DNA and become non-infectious prophages(lysogenic cycle). Nowadays, only strictly lytic phages are chosen forphage therapy.

Currently, several phage therapy clinical trials are in progress andthey all rely on a mixture of different purified phages, also calledphage cocktail. Usually, phage cocktails are composed of at least 3phages, up to 10 phages in some cases. Combining phages permit toincrease the host range of the drug by combining each individual phagehost range. Because the different phages have different host receptors,it also reduces the chance that, through mutation of a receptor,resistant bacterium arises. Phage cocktail is therefore a combination ofdifferent phages, each one of them encoding different proteins that willlead to the death of the host. It means that each phage can kill thecell in a different way (degrading DNA, bursting the cell, hijackingmolecular machinery, etc.). Moreover, each phage genome is contained indifferent phage particles targeting different receptors. This turns outto be regulatory difficult to get approved because of its complexity.

On the contrary, the use of packaged phagemids (viral particle wherephage genome is replaced by a plasmid of interest) allows to have adefined and control way of killing the host. Example of packagedphagemids encoding CRISPR-Cas9 or toxins have shown promising results inkilling targeted bacterial population (Bikard et al., 2012, Cell Host &Microbe 12, 177-186; Jiang et al., 2013, Nat Biotechnol 31, 233-239;Krom et al., 2015, Nano Letters 15, 4808-4813; Bikard et al, 2014, NatBiotech 11, Vol. 32, Citorik, R et al, 2014, Nat Biotech 11, Vol. 32).

However, the use of packaged phagemid has been restricted to a singleplasmid encapsidated in a single viral particle. This is not optimal forin vivo and clinical applications. Indeed, due to the genetic andepigenetic heterogeneity of pathogenic bacterial population in vitro andin vivo, the chance for a single viral particle to be injected in allclinically relevant strains is low. In addition, a single mutationchanging the receptor could be enough for a resistant bacterialpopulation to spread.

WO2008/157515 relates to the cloning of genomic librairies using phageencapsidation mechanisms. In particular, encapsidation initiation sites(PIS) are introduced at the level of a target DNA by transposition.These sites allow the encapsulation of the target DNA in phage capsids.The DNA encapsidated is then purified, ligated with a cohesive end andre-encapsidated and cloned into a bacterial strain for furthersequencing. More particularly, this document discloses a plasmid pDW7(FIG. 5) comprising the encapsidation sites cos and pac. However, thepurpose of WO2008/157515 has nothing in common with the purpose of thepresent invention. Indeed, the method described in this document willnever lead to the encapsidation in different delivery vehicles and pDW7has not been designed for and does not code for the expression of aprotein of interest into the target bacteria.

SUMMARY OF THE INVENTION

To overcome these problems, a first phagemid cocktail or mixture hasbeen developed based on a single payload containing at least 2orthogonal packaging sites, allowing its packaging into at least twodifferent bacterial delivery vehicles.

The invention concerns a pharmaceutical composition comprising at leasttwo different bacterial delivery vehicles into which the same payload ispackaged. Such payload comprises a nucleic acid sequence of interestunder the control of a promoter and at least two orthogonal bacterialvirus packaging sites that allow packaging of said payload into said atleast two different bacterial delivery vehicles.

Preferably, the at least two orthogonal bacterial virus packaging sitesare at least two different cos sites, at least two different pac sitesor at least two different concatemer junction sites.

Alternatively, the at least two orthogonal bacterial virus packagingsites are at least one cos site and at least one pac site, at least onecos site and at least one concatemer junction site, at least one pacsite and at least one concatemer junction site, or at least one cossite, at least one pac site and at least one concatemer junction site.

In one aspect, the at least two orthogonal bacterial virus packagingsites are selected in the group consisting of λ cos site, P4 cos site,SPP1 pac site, P1 pac site, T1 pac site, mu pac site, P22 pac site, φ8pac site, Sf6 pac site, 149 pac site, T7 concatemer junction, and A1122-concatemer junction. For instance, the at least two orthogonalbacterial virus packaging sites comprising λ cos site and P4 cos site,or λ cos site, P4 cos site and P1 pac site, or λ cos site, P4 cos siteand T7 concatemer junction, or λ cos site, P4 cos site, P1 pac site andT7 concatemer junction.

The nucleic sequence of interest of the payload according to theinvention can be selected from the group consisting of a Cas nuclease, aCas9 nuclease, a guide RNA, a single guide RNA (sgRNA), a CRISPR locus,a toxin, a gene expressing an enzyme such as a nuclease or a kinase, aTALEN, a ZFN, a meganuclease, a recombinase, a bacterial receptor, amembrane protein, a structural protein, a secreted protein, a geneexpressing resistance to an antibiotic or to a drug in general, a geneexpressing a toxic protein or a toxic factor, and a gene expressing avirulence protein or a virulence factor, or any combination thereof.

The nucleic sequence of interest may particularly be an encodingelement(s) of the CRISPR/Cas system for the reduction of gene expressionor inactivation of a gene selected from the group consisting of anantibiotic resistance gene, virulence factor or protein gene, toxinfactor or protein gene, a gene expressing a bacterial receptor, amembrane protein, a structural protein, a secreted protein, and a drugresistance gene, or any of their combination thereof.

The bacterial delivery vehicles according to the invention canparticularly be bacterial viruses, preferably bacterial viruses selectedfrom the group consisting of BW73, B278, D6, D108, E, E1, E24, E41,FI-2, FI-4, FI-5, HI8A, Ff18B, i, MM, Mu, 025, PhI-5, Pk, PSP3, P1, P1D,P2, P4, S1, Wφ, φK13, φ1, φ2, φ7, φ92, 7 A, 8φ, 9φ, 18, 28-1, 186, 299,HH-Escherichia (2), AB48, CM, C4, C16, DD-VI, E4, E7, E28, FI1, FI3, H,H1, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7, Ox-I, Ox-2,Ox-3, Ox-4, Ox-5, Ox-6, PhI-I, RB42, RB43, RB49, RB69, S, SaI-I, Sa1-2,Sa1-3, Sa1-4, Sa1-5, Sa1-6, TC23, TC45, TuII*-6, TuIP-24, TuII*46,TuIP-60, T2, T4, T6, T35, al, 1, IA, 3, 3A, 3T+, 5φ, 9266Q, CFO103,HK620, J, K, K1F, m59, no. A, no. E, no. 3, no. 9, N4, sd, T3, T7, WPK,W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, Φ04-CF, Φ05, Φ06, Φ07, φ1, φ1.2,φ20, φ95, φ263, φ1O92, φ1, φ11, Ω8, 1, 3, 7, 8, 26, 27, 28-2, 29, 30,31, 32, 38, 39, 42, 933W, NN-Escherichia (1), Esc-7-11, AC30, CVX-5, C1,DDUP, EC1, EC2, E21, E29, F1, F26S, F27S, Hi, HK022, HK97, HK139, HK253,HK256, K7, ND-I, PA-2, q, S2, T1,), T3C, T5, UC-I, w, β4, γ2, λ, ΦD326,φγ, Φ06, Φ7, Φ10, φ80, χ, 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000,AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, K1O, ZG/3A, 5, 5A,21EL, H19-J and 933H.

Particularly, the bacterial delivery vehicles are capable of targetingat least two different bacteria and of introducing the payload into thebacteria.

Alternatively, the bacterial delivery vehicles are capable of targetingthe same bacteria and of introducing the payload into these bacteria.

The pharmaceutical composition according to the invention comprise thedisclosed bacterial delivery vehicles and at least one additional activeingredient, for instance a prebiotic and/or a probiotic and/or anantibiotic, and/or another antibacterial or antibiofilm agent, and/orany agent enhancing the targeting of the bacterial delivery vehicle to abacteria and/or the delivery of the payload into a bacteria.

The pharmaceutical composition according to the invention may be for useas a medicament.

In a particular embodiment, the pharmaceutical composition may be usedfor in-situ bacterial production of a compound of interest, preferablysaid compound of interest being produced inside the targeted bacteria,secreted from the targeted bacteria or expressed on the surface of thetargeted bacteria. Particularly, the compound of interest can be anantigen expressed on the surface of the targeted bacteria forprophylactic and/or therapeutic vaccination.

The pharmaceutical composition according to the invention may be for usein the treatment of a disorder or disease caused by a bacterium,preferably by an antibiotic-resistant bacterium, such as an infection,preferably a bacterial infection, inflammatory diseases, auto-immunediseases, cancers, metabolic disorders and/or brain disorders.

The pharmaceutical composition according to the invention may also befor use in the prevention of a disorder or a disease caused by abacterium found in a subject, preferably by an antibiotic-resistantbacterium, such as an infection, preferably a bacterial infection,inflammatory diseases, auto-immune diseases, cancers, metabolicdisorders and/or brain disorders.

The invention also concerns a payload comprising a nucleic acid sequenceof interest under the control of a promoter and at least two orthogonalbacterial virus packaging sites that allow packaging of the payload intosaid at least two different bacterial delivery vehicles. Such payloadmay be a plasmid.

The invention finally concerns a bacterial delivery vehicle comprisingthe payload according to the invention. Such bacterial delivery vehiclemay be a bacterial virus particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Concentration of phagemid particles produce from the differentlysogenes.

FIG. 2: Data from three independent phagemids production.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention relates to a payload suitable to be packaged intoat least two different bacterial delivery vehicles. This payloadcomprises at least two orthogonal bacterial viruses packaging sites thatallow packaging of the payload into at least two different bacterialdelivery vehicles. Indeed, the advantage is to be able to deliver thesame payload to target cells by at least two different bacterialdelivery vehicles. Then, it allows the preparation of a pharmaceuticalor veterinary composition comprising at least two different bacterialdelivery vehicles, each bacterial delivery vehicle having the samepayload. In addition, depending on the patient or subject conditions,the person skilled in the art will be able to select the mostappropriate bacterial delivery vehicle for each case and to prepare theselected bacterial delivery vehicle with the packaged payload.

The present disclosure relates to a bacterial delivery vehiclecomprising such a payload packaged into the bacterial delivery vehicle.It relates to a composition, especially a pharmaceutical or veterinarycomposition, comprising at least two different bacterial deliveryvehicles, the bacterial delivery vehicles comprising the same payload.

It also relates to a bacterial cell comprising such a payload, inparticular bacterial cells capable of producing a bacterial deliveryvehicle comprising such a payload, and to a method for producing such abacterial delivery vehicle.

The present disclosure relates to the use of the bacterial deliveryvehicle comprising such a payload or the composition, especially apharmaceutical or veterinary composition, comprising at least twodifferent bacterial delivery vehicles, the bacterial delivery vehiclescomprising the same payload as a medicament, especially in the treatmentof a disorder or disease, in particular caused by a bacterium.

In another aspect a kit is provided comprising a payload as definedherein, optionally a satellite phage and/or a helper phage to promotethe packaging of the payload in a delivery vehicle, such as a bacterialvirus particle, or the structural and functional proteins necessary topromote an in vitro packaging of the payload in a bacterial virusparticle, and optionally bacterial cells suitable for packaged payloadproduction.

The bacterial delivery vehicles, preferably the bacterial virusparticle, are prepared from bacterial virus, in particularbacteriophages. The bacterial viruses are chosen in order to be able tointroduce the payload into the targeted bacteria.

Definitions

To facilitate the understanding of the invention, a number of terms aredefined below.

The terms “polynucleotide”, “nucleic acid” and “nucleic acid sequence”are equivalent and refer to a polymeric form of nucleotide of anylength, preferably to a sequence of at least two nucleotides covalentlylinked together which can be single-stranded or double-stranded orcontains portion of both single-stranded and double-stranded sequence.Nucleic acids (e.g., components, or portions of the nucleic acids) ofthe present invention may be naturally occurring or engineered.Engineered nucleic acids include recombinant nucleic acids and syntheticnucleic acids. Nucleic acids can be in the form of a circular sequenceor a linear sequence or a combination of both forms. The nucleic acidcan be DNA, both genomic or cDNA, or RNA or a combination of both. Thenucleic acid may contain any combination of deoxyribonucleotides andribonucleotides, and any combination of bases. Any combination of theabove features of a nucleic acid is also encompassed by the presentinvention.

As used herein, the term “gene” can be a genomic gene comprisingtranscriptional and/or translational regulatory sequences and/or acoding region and/or non-translated sequences (e.g., introns, 5′- and3′-untranslated sequences and regulatory sequences). The coding regionof a gene can be a nucleotide sequence coding for an amino acid sequenceor a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA andantisense RNA. A gene can also be an mRNA or cDNA corresponding to thecoding regions (e.g. exons and miRNA) optionally comprising 5′- or 3′untranslated sequences linked thereto. A gene can also be an amplifiednucleic acid molecule produced in vitro comprising all or a part of thecoding region and/or 5′- or 3′-untranslated sequences linked thereto.

The terms “polypeptide” and “protein” are used interchangeably herein.Polypeptides described herein may be composed of standard amino acids(i.e., the 20 L-alpha-amino acids that are specified by the geneticcode, optionally further including selenocysteine and/or pyrrolysine).Polypeptides may comprise one or more non-standard amino acids.Non-standard amino acids can be amino acids that are found in naturallyoccurring polypeptides, e.g., as a result of post-translationalmodification, and/or amino acids that are not found in naturallyoccurring polypeptides. Polypeptides may comprise one or more amino acidanalogues known in the art. Beta-amino acids or D-amino acids may beused. One or more of the amino acids in a polypeptide or peptide may bemodified, for example, by the addition of a chemical entity such as acarbohydrate group, a phosphate group, a fatty acid group, a linker forconjugation, functionalization, etc. A polypeptide that has anon-polypeptide moiety covalently or non-covalently associated may stillbe referred to as a “polypeptide”. Polypeptides may be purified fromnatural sources, produced in vitro or in vivo in suitable expressionsystems using recombinant DNA technology, synthesized through chemicalmeans such as conventional solid phase peptide synthesis and/or usingmethods involving chemical ligation of synthesized peptides. The term“polypeptide sequence” or “protein sequence” or “amino acid sequence” asused herein can refer to the polypeptide material itself and/or to thesequence information (i.e. the succession of letters or three lettercodes used as abbreviations for amino acid names) that biochemicallycharacterizes a polypeptide.

The term “heterologous” in the context of a nucleic acid construct(payload, plasmid, vector or cargo) indicates that the nucleic acidcomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, a nucleic acid istypically recombinantly produced, has two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., anucleic acid encoding a fluorescent protein from one source and anucleic acid encoding a peptide sequence from another source. Similarly,a heterologous protein indicates that the protein comprises two or moresubsequences that are not found in the same relationship to each otherin nature. In the context of a host cell, it means that the sequenceencodes a protein which originates from a source different from the cellin which it is introduced or that the coding sequence comes from thesame species as the cell in which it is introduced but it is consideredheterologous due to its environment which is not natural, for examplebecause it is under the control of a promoter which is not its naturalpromoter, or is introduced at a location which differs from its naturallocation.

As used herein, the term «payload» refers to any nucleic acid sequencethat can be transferred into a bacterium by a bacterial deliveryvehicle. The term «payload» may particularly refer to a plasmid, vectoror cargo as defined hereafter. Preferably, the payload can be a phagemidor phasmid obtained from natural, evolved or engineered bacteriophagegenome. The payload can also be composed in part of phagemid or phasmidobtained from a natural, evolved or engineered bacteriophage genome.

As used herein, the term “same payload” or “identical payload” areequivalent and refer to bacterial delivery vehicles containing payloadwith the same nucleic acid sequence enconding biological functions, i.ephage packaging, plasmid replication, plasmid selection, expression ofproteins of interest in the target bacterium. In a first aspect, twopayloads are considered as “same payload” when they have exactly thesame nucleic acid sequence. In a second aspect, two payloads areconsidered as “same payload” when they encode the same biologicalfunctions. In this second aspect, the “same payload” may refer to twopayloads having at least 70, 80, 90 or 95% of identity between eachother. In other words, the “same payload” may refer to two payloadshaving at least 70, 80, 90 or 95% of identity between each other andencoding the same biological functions. In a particular aspect, thepayloads have the same sequence except the tracer DNA sequences.

As used herein, the terms “plasmid”, “vector” and “cargo” are equivalentand refer to a payload, such as DNA or RNA, transferred into a host cellusing a bacterial delivery vehicle. A vector may comprise an origin ofreplication, a selectable marker, and optionally a suitable site for theinsertion of a sequence or gene. A vector can be either aself-replicating extrachromosomal vector or a vector which integratesinto a host genome. It can also comprise expression elements including,for example, a promoter, the correct translation initiation sequencesuch as a ribosomal binding site and a start codon, a termination codon,and a transcription termination sequence. A plasmid may also compriseother regulatory regions such as enhancers, silencers and boundaryelements/insulators to direct the level of transcription of a givengene. Vectors capable of directing the expression of genes and/ornucleic acid sequence to which they are operatively linked can also bereferred to herein as “expression vectors”. There are several commontypes of vectors including plasmids, bacterial virus genomes, phagemids,virus genomes, cosmids, and artificial chromosomes. The plasmid can be avector for stable or transient expression of a gene or sequence. In oneembodiment, the plasmid of the invention is a phagemid or plasmid andrefers to a vector that derives from both a plasmid and a bacterialvirus genome. The plasmid may comprise a plasmid origin of replication(ori), a packaging signal and/or a tracer DNA sequence.

As used herein, the term “packaged payload” or “payload packaged into adelivery vehicle” refers to a payload which is contained into a deliveryvehicle to promote its delivery into the targeted bacteria.

As used herein, the term “packaged plasmid” or “plasmid packaged into abacterial virus particle” refers to a plasmid which is encapsidated intoa proteinaceous envelope or capsid of a bacterial virus.

As used herein, the term “bacterial delivery vehicle” refers to any meanthat allows the transfer of a payload into a cell, preferably abacterial cell. There are several types of delivery vehicle encompassedby the present invention including, without limitation, bacteriophagescaffold, virus scaffold, protein-based or peptide-based deliveryvehicle. Any combination of delivery vehicles is also encompassed by thepresent invention. The delivery vehicle can particularly refer to abacteriophage derived scaffold and can be obtained from a natural,evolved or engineered capsid. The bacterial delivery vehicle can also bea proteinaceous envelope or capsid. In an embodiment, the deliveryvehicle is the payload as bacteria are naturally competent to take up apayload from the environment on their own. In certain embodiments, thedelivery system can be administered to a subject in need thereof. Thebacterial cell can be an isolated cell (e.g. in a bacteria cell culture)or a cell associated with a subject in which inhibiting or promoting theexpression of a target gene or a sequence of interest is desired.

As used herein, the terms “bacterial virus”, “phage” or “bacteriophage”are used interchangeably and refer to a functional phage particlecomprising a nucleic acid packaged into a proteinaceous envelope orcapsid. The term also refers to portions of the bacterial virus,including, e.g., a head portion, or an assembly of phage components,which provide substantially the same functional activity.

As used herein, the terms “bacterial virus particle” or “virion” areequivalent and refer to a phage shape particle comprising a payload. Thebacterial virus particle can be a proteinaceous envelope or capsid.Particularly, it refers to a bacterial virus particle devoid of abacteriophage genome.

As used herein, the terms “proteinaceous envelope”, “capsid”, or “coatproteins” are equivalent and refer to the shape of the shell of proteinsthat protects the nucleic acid (i.e., genome) of a virus that isgenerally composed of structural units, or capsomers. Capsids arebroadly classified according to their structure and shape known by theperson skilled in the art. Preferably, they refer to bacteriophagecapsids or coat proteins.

As used herein, the terms “different bacterial delivery vehicle”,“different virus particles” or “distinct virus particles” or “differentvirus capsids” are equivalent and refer to the packaging of identicalplasmids into bacterial delivery vehicles such as viral particles whichare different, e.g. two different bacteriophage capsids.

As used herein, the terms “packaged” or “encapsulated” are equivalentand refer to the packaging of a payload, especially a plasmid, into abacterial delivery vehicle. In one embodiment, the term “packaging” maybe equivalent to the term “encapsidation” which refers to the packagingof a payload into a bacterial virus particle or capsid.

As used herein, the terms “orthogonal packaging sites” refer topackaging signals that are different and independent, meaning that theylead to separate packaging, i.e. into at least two different bacterialdelivery vehicles such as capsids or bacterial virus particles.

The term “helper phage” as used herein refers to a virus beingco-infected with a principal defective virus, in particular the payloadto be packaged into a bacterial virus particle. The helper phageprovides in trans the functions of which the first is deprived. In oneembodiment, the packaged payload according to the invention may beproduced using a helper phage strategy, well known to those skilled inthe art. In this embodiment, the helper phage comprises all the genescoding for the structural and functional proteins that are indispensablefor the payload according to the invention to be packaged orencapsidated (i.e. helper phage provides all the necessary gene productsfor particle formation). The helper phages are mutated wild-type phagecontaining a defective origin of replication or packaging signal, andhence, are inefficient in self-packaging, thus only bacterial virusparticles carrying the deliverable nucleic acid (i.e., the payload orplasmid) will be produced. Helper phages may be chosen so that theycannot induce lysis of the host used for the particle production. It isunderstood by one skilled in the art that some bacteriophages aredefective and need a helper phage for replication and/or packaging.Thus, according to the bacterial virus chosen in the present inventionto prepare the bacterial virus particles, one skilled in the art wouldknow if and which a helper phage is required.

As used herein, the terms “viral satellite genes” refers to genesderived from a satellite virus or satellite phage. Satellite phage arealso known as a subviral agent and are composed of nucleic acid thatdepends on the co-infection of a host cell with a helper virus for allthe morphogenetic functions, whereas for all its episomal functions(integration and immunity, multicopy plasmid replication) the satelliteis completely autonomous from the helper. In one embodiment, thesatellite genes can encode proteins that promote capsid size reductionof the helper phage, as described for the P4 Sid protein that controlsthe P2 capsid size to fit its smaller genome.

As used herein, the terms “promoter” and “transcriptional promoter” areequivalent and refer to a control region of a nucleic acid sequence atwhich transcription initiation and rate of transcription of theremainder of a nucleic acid sequence are controlled. A promoter may alsocontain sub-regions to which regulatory proteins and molecules may bind,such as RNA polymerase and other transcription factors. A promoterdrives transcription of the nucleic acid sequence that it regulates.Herein, a promoter is considered to be “operably linked” when it is in acorrect functional location and orientation in relation to a nucleicacid sequence it regulates to control (“drive”) transcriptionalinitiation of that sequence.

As used herein, the term “origin of replication” refers to a particularsequence in a genome at which replication is initiated. This can eitherinvolve the replication of DNA in living organisms such as prokaryotesand eukaryotes, or that of DNA or RNA in viruses. Preferably, it refersto a bacterial origin of replication or a phage origin of replicationthat is present on the plasmid according to the invention.

As used herein, the term “selection marker” refers to a gene which isused to confirm the cloning of a gene or to confirm or ensure thepresence of a plasmid in a bacterium. The selection marker can be amarker gene providing selectable phenotypes such as drug resistance,auxotrophy, resistance to cytotoxic agents, or surface proteinexpression. For example, an antibiotic-resistant gene, a gene allowingto overcome auxotrophy, a color-developing enzyme gene or aluminescent/fluorescent gene may be used. This confers a “selectiveadvantage” to bacteria carrying such selection marker so as to be ableto grow on medium supplied with antibiotics, heavy metals, or on mediumwithout essential component such as amino acid.

As used herein, the term “inactivation” refers to the direct or indirectinhibition or decrease of the expression of a gene, or of the biologicalfunction of the protein, or of the production of specific gene products(protein or RNA), compared to a normal or previous condition. Theregulation of the gene expression can be on the gene itself (i.e.cleavage, modifications), at the stage of transcription (i.e. usingsilencers or repressors), or using RNAi (e.g. siRNA, shRNA, endogenousmicroRNA or artificial microRNA), TALEN, ZFN, meganuclease or CRISPR/Cassystem. In one embodiment, the CRISPR/Cas9 system is used to inactivategene expression such as an antibiotic resistance gene, a virulence geneor a toxin gene present in the targeted bacteria.

As used herein, the term “bacterium” or “bacteria” refers to anyprokaryotic microorganisms that exist as a single cell or in a clusteror aggregate of single cells. The term “bacterium” encompasses allvariants of bacteria (e.g., endogenous bacteria, which naturally residein a closed system, environmental bacteria or bacteria released forbioremediation or other efforts). Bacteria of the present disclosureinclude bacterial subdivisions of Eubacteria and Archaebacteria.Eubacteria can be further subdivided into Gram-positive andGram-negative Eubacteria. Also included herein are those classifiedbased on gross morphology alone (e.g., cocci, bacilli). In someembodiments, the bacteria are Gram-negative cells, and in otherembodiments, the bacteria are Gram-positive cells.

As used herein, the terms “targeted bacteria” refers to the bacteriagender, species or strains that can be recognized by the bacterialdelivery vehicle according to the invention and in which the bacterialdelivery vehicle promote the introduction of the payload into saidtargeted bacteria. The specific spectrum of bacteriophages is known bythe person skilled in the art, so that the person skilled in the artwould know what would be the targeted bacteria according to the chosenphage. Preferably, the targeted bacteria are bacteria present in thehuman body (i.e., bacteria of the microbiota). Even more preferably, thetargeted bacteria are bacteria presenting specific phenotypicalcharacteristics of interest, such as but not limited toantibiotic-resistance.

As used herein, the terms “different bacteria” can refer to distinctbacteria species or can also refer to different strains or geneticvariants or subtypes or genotypes of bacteria.

As used herein, the terms “containing the same payload” refers to thepayload content of different bacterial delivery vehicles, meaning thatidentical payloads are packaged and contained into at least twodifferent bacterial delivery vehicles. In an embodiment where thebacterial delivery vehicle is a bacterial virus, these terms mean thatidentical payloads are encapsidated and contained in at least twodifferent bacterial virus capsids or particles (i.e., each bacteriophagehas encapsidated and contains distinct copies of the same plasmid whichpresent the same properties).

As used herein, the terms “antibiotic” and “antibacterial” areequivalent and refer to a type of antimicrobial active ingredient usedin the treatment and prevention of bacterial infections. It can be aclassical antibiotic that is produced by a microorganism that isantagonistic to the growth of other microorganisms and also encompassesmore generally an antimicrobial agent that is capable of killing orinhibiting the growth of a microorganism, including chemicallysynthesized versions and variants of naturally occurring antibiotics.

As used herein, the term “antibiotic resistance gene” encompasses agene, or the encoding portion thereof, which encodes a product ortranscribes a functional RNA that confers antibiotic resistance. Theantibiotic resistance gene may for example encode an enzyme whichdegrades an antibiotic, or an enzyme which modifies an antibiotic, or apump such as an efflux pump, or a mutated target which suppresses theeffect of the antibiotic.

As used herein, the terms “antibiotic resistant bacteria” refer to theability of a bacterium to resist the effects of medication used againstthem.

The term “treatment” refers to any act intended to ameliorate the healthstatus of patients or subjects such as therapy, prevention, prophylaxisand retardation of the infection. It designates both a curativetreatment and/or a prophylactic treatment of a disease. A curativetreatment is defined as a treatment resulting in cure or a treatmentalleviating, improving and/or eliminating, reducing and/or stabilizingthe symptoms of a disease or the suffering that it causes directly orindirectly. A prophylactic treatment comprises both a treatmentresulting in the prevention of a disease and a treatment reducing and/ordelaying the incidence of a disease or the risk of its occurrence. Incertain embodiments, such term refers to the improvement or eradicationof a disease, a disorder, an infection or symptoms associated with it.In other embodiments, this term refers to minimizing the spread or theworsening of an infection; e.g., resulting from antibiotic-resistantbacteria.

As used herein, the term “disorder” refers to an incorrectly functioningorgan, part, structure, or system of the body. Preferably, the termdisorder refers to a health disorder e.g. an illness that disruptsnormal physical or mental functions. More preferably, the term disorderrefers to a bacterial disease that is caused by or associated withbacteria or bacterial components that affect animals and/or humans. In aparticular embodiment, the term disorder refers to the consequences of abacterial infection, preferably by antibiotic resistant bacteria, or ofa dysbiosis.

As used herein, the term “disease” refers to a disordered or incorrectlyfunctioning organ, part, structure, or system of the body resulting fromthe effect of genetic or developmental errors, infection, poisons,nutritional deficiency or imbalance, toxicity, or unfavorableenvironmental factors. Preferably, the term disease refers to abacterial disease that is caused by bacteria or bacterial componentsthat affect animals and/or humans. In a particular embodiment, the termdisease refers to the consequences of a bacterial infection, preferablyby antibiotic resistant bacteria, or of a dysbiosis.

As used herein, a “pharmaceutical or veterinary composition” refers to apreparation of one or more of the active agents, such as the bacterialdelivery vehicles containing the payload according to the invention,with optional other chemical components such as physiologically suitablecarriers and excipients. The purpose of a pharmaceutical or veterinarycomposition is to facilitate administration of the active agent to anorganism. Compositions of the present invention can be in a formsuitable for any conventional route of administration or use. In oneembodiment, the pharmaceutical or veterinary composition furthercomprises a pharmaceutically or veterinary acceptable vehicle.

A “pharmaceutically or veterinary acceptable vehicle” as referred toherein, is any known compound or combination of compounds that are knownto those skilled in the art to be useful in formulating pharmaceuticalor veterinary compositions. The pharmaceutically or veterinaryacceptable vehicle may be a solid, and the composition may be in theform of a powder or tablet. The vehicle may also be an encapsulatingmaterial. In powders, the vehicle is a finely divided solid that is inadmixture with the finely divided active agents according to theinvention. In tablets, the active agent (e.g. the particle or system ofthe invention) may be mixed with a vehicle having the necessarycompression properties in suitable proportions and compacted in theshape and size desired. The powders and tablets preferably contain up to99% of the active agents. The pharmaceutical or veterinary vehicle maybe a gel and the composition may be in the form of a cream or the like.However, the pharmaceutical or veterinary vehicle may alternatively be aliquid, and the pharmaceutical or veterinary composition is in the formof a solution. Liquid vehicles are used in preparing solutions,suspensions, emulsions, syrups, elixirs and pressurized compositions.Sterile liquid vehicles are useful in sterile liquid form compositionsfor enteral administration.

As used herein, a “prebiotic” refers to an ingredient that allowsspecific changes, both in the composition and/or activity in thegastrointestinal microbiota that may confer benefits upon the host. Aprebiotic can be a comestible food or beverage or ingredient thereof. Aprebiotic may be a selectively fermented ingredient. Prebiotics mayinclude complex carbohydrates, amino acids, peptides, minerals, or otheressential nutritional components for the survival of the bacterialcomposition.

As use herein, the term “probiotic” refers to a dietary supplement basedon living microbes which, when taken in adequate quantitis, has abeneficial effect on the host organism by strengthening the intestinalecosystem. A probiotic can comprise a non-pathogenic bacterial or fungalpopulation, e.g., an immunomodulatory bacterial population, such as ananti-inflammatory bacterial population, with or without one or moreprebiotics. They contain a sufficiently high number of living and activeprobiotic microorganisms that can exert a balancing action on gut floraby direct colonisation. It must be noted that, for purposes of thepresent description, the term “probiotic” is taken to mean anybiologically active form of probiotic, preferably but not limited tolactobacilli, bifidobacteria, streptococci, enterococci,propionibacteria or saccaromycetes but even other microorganisms makingup the normal gut flora, or also fragments of the bacterial wall or ofthe DNA of these microorganisms. These compositions are advantageous inbeing suitable for safe administration to humans and other mammaliansubjects and are efficacious for the treatment, prevention, of abacterial infection.

A “therapeutically effective amount” is an amount which, whenadministered to a subject, is the amount of active agent that is neededto treat the targeted disease or disorder, or to produce the desiredeffect, e.g. result in effective delivery of the bacterial deliveryvehicles containing the payload to the targeted bacteria.

As used herein, the term “subject” or “patient” refers to an animal,preferably to a mammal, even more preferably to a human, including adultand child. However, the term “subject” also encompasses non-humananimals, in particular mammals such as dogs, cats, horses, cows, pigs,sheep and non-human primates, among others.

The term “percentage of identity” in relation to sequences designatesthe level of identity or homology between said sequences and may bedetermined by techniques known per se in the art. Typically, thepercentage of identity between two nucleic acid sequences is determinedby means of computer programs such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1996, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman, S. B. and Wunsch, C.D., (1970), Journal of MolecularBiology, 48, 443-453). With settings adjusted to e.g., DNA sequences(particularly: GAP creation penalty of 5.0 and GAP extension penalty of0.3), nucleic acid molecules may be aligned to each other using thePileup alignment software available as part of the GCG program package.For comparing two amino acid sequences, one can use, for example, thetool “Emboss needle” for pairwise sequence alignment of proteinsproviding by EMBL-EBI and available on: ebi.ac.uk using defaultsettings: (I) Matrix: BLOSUM62, (ii) Gap open: 10, (iii) gap extend:0.5, (iv) output format: pair, (v) end gap penalty: false, (vi) end gapopen: 10, (vii) end gap extend: 0.5.

Sequence identity between nucleotide or amino acid sequences can bedetermined by comparing an alignment of the sequences. When anequivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position.Scoring an alignment as a percentage of identity is a function of thenumber of identical amino acids or bases at positions shared by thecompared sequences. When comparing sequences, optimal alignments mayrequire gaps to be introduced into one or more of the sequences to takeinto consideration possible insertions and deletions in the sequences.Sequence comparison methods may employ gap penalties so that, for thesame number of identical molecules in sequences being compared, asequence alignment with as few gaps as possible, reflecting higherrelatedness between the two compared sequences, will achieve a higherscore than one with many gaps. Calculation of maximum percent identityinvolves the production of an optimal alignment, taking intoconsideration gap penalties.

As used herein, the term “compatible size range” or “size requirement”refers to the size (kilobase) of a payload as disclosed herein that willbe differentially packaged into the at least two different bacterialdelivery vehicles. It is known by the person skilled in the art that,for identical payloads to be packaged into different capsids, they needto be a suitable substrate for each packaging mechanisms (e.g. headfulor cohesive packaging mechanisms) and meet the size requirement to fitin the cavity of either of the at least two different bacterial viruscapsids.

As used herein, “PFU” means plaque forming unit, as it is well definedin the art. Lytic bacteria viruses lyse the host cell, causing a zone ofclearing (or plaque) on a culture plate. Theoretically, each plaque isformed by one phage and the number of plaques multiplied by the dilutionfactor is equal to the total number of phages in a test preparation.

As used herein, “CFU” means colony forming unit, as it is well definedin the art. This unit is used to estimate the number of viable bacteriaor yeast in a sample, and refers to a mass of bacterial cells or yeastcells from the same bacterial or yeast progenitor.

“In vitro” refers to procedures that are performed outside of a cell.For example, purified enzymes or extracts of cells can be used toperform procedures in a vessel, such as a test tube.

“Ex vivo” refers to procedures that are performed outside of amulticellular organism, but use whole cells. For example, live cellsfrom a subject, such as a human, can be cultured outside of the body andthese cells can be used in testing procedures.

“In vivo” refers to procedures that are performed on a whole organism,such as a subject, including a human, such as in clinical trials. Invivo procedures can also be performed on non-human subjects, such asanimal models.

The term “and/or” as used herein is to be taken as specific disclosureof each of the two specified features or components with or without theother. For example “A and/or B” is to be taken as specific disclosure ofeach of (i) A, (ii) B and (iii) A and B, just as if each is set outindividually.

The term “a” or “an” can refer to one of or a plurality of the elementsit modifies (e.g., “a reagent” can mean one or more reagents) unless itis contextually clear either one of the elements or more than one of theelements is described.

The term “about” as used herein in connection with any and all values(including lower and upper ends of numerical ranges) means any valuehaving an acceptable range of deviation of up to +/−10% (e.g., +/−0.5%,+/−1%, +/−1.5%, +/−2%, +/−2.5%, +/−3%, +/−3.5%, +/−4%, +/−4.5%, +/−5%,+/−5.5%, +/−6%, +/−6.5%, +/−7%, +/−7.5%, +/−8%, +/−8.5%, +/−9%,+/−9.5%). The use of the term “about” at the beginning of a string ofvalues modifies each of the values (i.e. “about 1, 2 and 3” refers toabout 1, about 2 and about 3). Further, when a listing of values isdescribed herein (e.g. about 50%, 60%, 70%, 80%, 85% or 86%) the listingincludes all intermediate and fractional values thereof (e.g., 54%,85.4%).

Payload of the Present Invention

The present invention relates to a payload suitable to be packaged intoat least two different bacterial delivery vehicles and comprising atleast two orthogonal bacterial viruses packaging sites.

To reproduce and survive, bacteria viruses need to package their genomeinside their capsids. For this purpose, they evolved differentsophisticated mechanisms differentiated by the nature of the phagetermini: (i) single-stranded cohesive ends, (ii) circularly permuteddirect terminal repeats, (iii) short, several hundred base pairs exact(non-permuted) direct terminal repeats, (iv) long, several thousand basepairs exact (non-permuted) direct terminal repeats, (v) terminal hostDNA sequences, and (vi) covalently bound terminal proteins.

Two major packaging mechanisms relying on two different types of terminican be used according to the present invention which permit payloadpackaging into at least two different bacterial delivery vehicles aredescribed hereafter. Two different packaging sites using the samemechanism and relying on different terminases can also be used accordingto the present invention.

In an aspect of the disclosure, the at least two packaging sites are notcomprised between a pair of transposable ends of a transposable element,especially Tn5 mosaic ends. In a very particular embodiment, the payloaddoes not comprise any transposable end and/or transposable element.

Alternatively, the payload may also be the bacterial delivery vehicle asbacteria are naturally competent to take up a payload from theenvironment on their own.

Headful Packaging (Circularly Permuted Direct Terminal Repeats)

A headful packing system may feed the nucleic acid into the cavity of aphage prohead in a linear processive manner causing the head to expanduntil it reaches a limit where the DNA inside exerts pressure againstthe inner wall sufficient to stop progression. This may induce aconformational change in the head, which activates endonucleolyticcleavage of incoming DNA opening the way for attachment of phage tailsto make infectious particles. Full heads may contain DNA moleculeswithin a narrow size range. The capacity of the capsids may set amaximum size limitation on the packaged DNA. Representative headfulpackaging systems include, but are not limited to, P1, P7, T4, KVP40,P22 and (1)29.

Single-Stranded Cohesive Ends Packaging

The packaging machinery may use a specific site to initiate andterminate packaging (cos). It may employ highly specific cos sites toinitiate and terminate packaging. These sites are cut by terminaseleaving base overhangs (cos L and cos R) at the ends of the packagedDNA. In λ's natural rolling circle packaging substrate, cos sites arespaced closer together than the full packaging limit determined by headsize. As a result, cos site spacing, and not head capacity, normallydetermines the length of virion DNA.

In one embodiment, the payload as disclosed hereafter can be packaged inthe at least two different bacterial delivery vehicles by headful and/orcohesive packaging mechanisms.

The payload of the present invention is able to be packaged into atleast two different bacterial delivery vehicles. This payload comprises:

-   -   at least two orthogonal bacterial virus packaging sites that        allow packaging into at least two different bacterial delivery        vehicles;    -   optionally, an origin of replication, preferably inactive in the        bacteria targeted by the at least two different bacterial        delivery vehicles;    -   a nucleic acid sequence of interest under the control of a        promoter.

Each of these features is described hereafter.

Payload Size

The size of the payload is selected so as to be suitable with thepackaging into the considered bacterial delivery vehicles. Similarly,the considered bacterial delivery vehicles can be selected for beingcompatible, in particular having a size suitable with the payload to bepackaged into such bacterial delivery vehicles.

In some embodiments, the payload disclosed herein comprises a size rangeof at least 100 base pairs (bp), at least 1 kilobase (kb), at least 2kilobases (kb), at least 3 kilobases (kb), at least 4 kb, at least 5 kb,at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb,at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least75 kb, at least 80 kb, at least 85 kb, at least 90 kb, at least 95 kb,at least 100 kb, at least 105 kb, at least 110 kb, at least 115 kb, atleast 120 kb, at least 125 kb, at least 130 kb, at least 135 kb, atleast 140 kb, at least 145 kb, at least 150 kb, at least 175 kb, atleast 200 kb, at least 225 kb, at least 250 kb, at least 275 kb, atleast 300 kb, at least 325 kb, at least 350 kb, at least 375 kb, atleast 400 kb, at least 425 kb, at least 450 kb, at least 475 kb, or atleast 500 kb of nucleic acids.

Bacterial Virus Packaging Sites

For successful packaging into a bacterial delivery vehicle, the payloadaccording to the invention comprises at least two different packagingsignal sequences. For the same payload to be packaged into differentcapsids, it should to be a suitable substrate for each packagingmechanisms (i.e. headful or cohesive packaging mechanisms). Thisincludes size requirement, no inhibition between packaging sites, andorigin of replication compatible with both packaging systems.

Packaging sites include but are not limited to SPP1 (SPP1 pac site), P1(P1 pac site), T1 (T1 pac site), T7 (T7 concatemer junction), lambda (λcos site), P4 (P4 cos site), mu (mu pac site), P22 (P22 pac site), φ8(φ8 pac site), Sf6 (Sf6 pac site), 149 (149 pac site), and A1 122 (A1122-concatamer junction). Other types of packaging sites include HK97packaging site, mEp235 packaging site, mEp043 packaging site, mEp234packaging site, mEp505 packaging site, mEp506 packaging site, mEpX1packaging site, mEpX2 packaging site, mEp390 packaging site, mEp460packaging site, mEp213 packaging site, mEp237 packaging site, HK022packaging site and phi80 packaging site. For most bacterial viruses, thepackaging site is termed the pac site. In some cases, the packaging siteis referred to as a concatemer junction (e.g. T7 concatemer junction).In every case, the packaging site is substantially isolated fromsequences naturally occurring adjacent thereto in the bacteria virusgenome.

For some bacterial viruses, the packaging site may be unknown. In thesecases, pac sites can be determined by taking advantage of the propertythat plasmids containing a functional bacterial virus pac site arepackaged. For example, the DNA sequences necessary for packaging bybacterial virus λ were determined by incorporating small restrictionfragments of the)\, phage genomic DNA into a plasmid (Hohn, B 1983 PNASUSA 80:7456-7460). Using a similar strategy, the pac or cos sites for anumber of bacterial viruses have been determined: λ (Miwa, T 1982 Gene20:267-279); Mu (Groenen, MA and van de Putte, P 1985 Virology144:520-522); filamentous bacteria viruses including fl, fd, M13, andIke (Russel, M and Model, P 1989 J Virol 1989 63:3284-3295); P22 (Petri,J B and Schmieger, H 1990 Gene 88:47-55; Wu, H et al. 2002 MolecMicrobiol 45:1631-1646); T7 (Chung, Y B and Hinkle, D C 1990 J Mo/Biol216:927-938), and T3 (Hashimoto, C and Fujisawa, H 1992 Virology187:788-795). The determination of DNA packaging strategy was alsoinvestigated by analysis of the terminase amino acid sequence oftailed-bacteriophage virions (Casj ens and Gillcrease, Methods Mol Biol.2009; 502: 91-111). A method to determine packaging sites and packagingmechanisms of phages using high-throughput sequencing data was describedrecently (Garneau J R, Depardieu F, Fortier L C, Bikard D and Monot M,Scientific Reports 2017 7(1):8292).

The payload according to the invention comprises at least two orthogonalpackaging sites, preferably two orthogonal bacterial virus packagingsites, that allow packaging of the payload into at least two differentbacterial delivery vehicles selected in the group consisting of pacsites, cos sites and concatemer junction sites or any other packagingsites with a different or unknown packaging mechanism or any combinationthereof.

In one embodiment, the at least two different packaging sites can be atleast two different cos sites (for example λ and P4 cos sites), at leasttwo different pac sites (for example Mu, P1 and P22 pac sites) or atleast two different concatemer junction sites (for example T7 and AI 122concatemer junctions).

In another embodiment, the at least two different packaging sites can beat least one cos site and at least one pac site (for example λ cos siteand P22 pac site), at least one cos site and at least one concatemerjunction site (for example λ cos site and T7 concatemer junction), atleast one pac site and at least one concatemer junction site (forexample P22 pac site and T7 concatemer junction), or at least one cossite, at least one pac site and at least one concatemer junction site(for example λ cos site, P22 pac site and T7 concatemer junction).

Particularly, the payload according to the invention comprises at leasttwo different packaging sites which can be selected in the groupconsisting of λ cos site, P4 cos site, SPP1 pac site, P1 pac site, T1pac site, mu pac site, P22 pac site, φ8 pac site, Sf6 pac site, 149 pacsite, T7 concatemer junction, A1 122-concatemer junction.

Preferably, the payload according to the invention comprises at leasttwo different packaging sites which are λ cos site and P4 cos site.Alternatively, the payload according to the invention comprises λ cossite and P4 cos site, or λ cos site, P4 cos site and P1 pac site, or λcos site, P4 cos site and T7 concatemer junction, or λ cos site, P4 cossite, P1 pac site and T7 concatemer junction.

In one embodiment, the payload according to the invention comprises atleast two different packaging sites which are selected in the table 1below. Preferably, the at least two different packaging sites areselected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 and any combinationthereof.

Particularly, the payload according to the invention comprises apackaging site of SEQ ID No. 3 and at least one different packaging siteselected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 4, SEQ ID No. 5 and SEQ ID No. 6.

In a particular embodiment, the payload according to the inventioncomprises at least three different packaging sites. Preferably the atleast three packaging sites are selected from the group consisting ofSEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 andSEQ ID No. 6. Particularly, the payload according to the inventioncomprises three packaging sites of SEQ ID No. 1, SEQ ID No. 3 and SEQ.ID No. 5, respectively.

In a yet further embodiment, the at least two or at least threedifferent packaging sites have at least 70% sequence identity, at least75% sequence identity, at least 80% sequence identity, at least 85%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity with any of the of the SEQ ID No. 1, SEQ ID No. 2, SEQ ID No.3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

By different packaging sites, it is meant packaging sites having lessthan 80% of sequence identity between each other's, less than 75% ofsequence identity, less than 70% of sequence identity, less than 65% ofsequence identity, less than 60% of sequence identity, less than 55% ofsequence identity, less than 50% of sequence identity, less than 45% ofsequence identity, less than 40% of sequence identity, less than 35% ofsequence identity, less than 30% of sequence identity, less than 25% ofsequence identity, less than 20% of sequence identity, less than 15% ofsequence identity, or less than 10% of sequence identity.

TABLE 1 Packaging sites DNA sequences SEQ ID Name Sequence 1 P1 PacACCTGGGACGATCACAAGAAGAATTTTGCTCGCCTGGCGCGAGATGGTGGTTACACCATCGCACAGTATGCCGCCGAGTTTAATCTTAACCCTAATACCGCACGTCGTTATCTCCGTGCCTTCAAAGAGGACACCAGGACTACGGACAGCCGCAAGCCAAATAAGCCAGTCAGGAAGCCACTAAAAAGCATGATCATTGATCACTCTAATGATCAACATGCAGGTGATCACATTGCGGCTGAAATAGCGGAAAAACAAAGAGTTAATGCCGTTGTCAGTGCCGCAGTCGAGAATGCGAAGCGCCAAAATAAGCGCATAAATGATCGTTCAGATGATCATGACGTGATCACCCGCGCCCACCGGACCTTACGTGATCGCCTGGAACGCGACACCCTGGATGATGATGGTGAACGCTTTGAGTTCGAAGTTGGCGATTACCTGATAGATAACGTTGAAGCGCGGAAGGCCGCGCGCGCTATGTTGCGTCGGTCCGGGGCCGATGTTCTGGAAACCACTCTTCTGGAAAAGTCTCTTTCTCATCTCCTTATGCTGGAGAACGCCAGGGATACGTGTATTCGCCTGGTGCAGGAAATGCGCGATCAGCAAAAAGACGATGATGAAGGTACTCCGCCTGAATACCGTATCGCGAGCATGCTAAACAGCTGTTCCGCGCAGATAAGCAGCCTGATCAACACCATTTACAGCATCCGGAATAACTATCGAAAAGAAAGCCGGGAGGCGGAAAAGCACGCTTTATCTATGGGGCAAGCTGGCATTG TTAAGCTGGCATA 2 Cos P4GCATGCGTTTTCCTGCCTCATTTTCTGCAAACCGCGCCATTCCCGGCGCGGTCTGAGCGTGTCAGTGCAACTGCATTAAAACCGCCCCGCAAAGCGGGCGGGCGAGGCGGGGAAAGCACCGCGCGCAAACCGACAAGTTAGTTAATTATTTGTGTAGTCAAAGTGCCTTCAGTACATACCTCGTTAATACATTGGAGCATAATGAAGAAAATCTATGGCCTATGGTCCAAAACTGTCTTTTTTGATGGCACTATCCTGAAAAATATGCAAAAAATAGATTGATGTAAGGTGGTTCTTGTCAGTGTCGCAAGATC CTTAAGAATTC 3 Cos lambdaCCAAAAAGCCTCGCTTTCAGCACCTGTCGTTTCCTTTCTTTTCAGAGGGTATTTTAAATAAAAACATTAAGTTATGACGAAGAAGAACGGAAACGCCTTAAACCGGAAAATTTTCATAAATAGCGAAAACCCGCGAGGTCGCCGCCCCGTAACCTGTCGGATCACCGGAAAGGACCCGTAAAGTGATAATGATTAT CATCTACATATCACAACGTGCGTAAAGG 4Cos P2 TAAGGTGCATTAAAACCGCCCCGTGAAGCGGGCGGGCGAGGCGGGGAAAGCACGGCGAGGCGGGGAAAGCACTGCGC GCTGACG 5 Cos 186TGTTTTGCATGCGTCAGGCTTGCCCGTTCTGGTTGTGCGTCGCCAGAGCTGGCGCGGCTCCAGAGTGGTCATGCAACTGCATTAAAACCGACCCATAAAGTGGGCAGGCGTGGCGGG GAAAGCATTGCGCGCCAGAGGTG 6 T7AGTCCATGCAGTTGGATTCCGTTAAGGTCGAGGGTGAAG concatemerTACTTGCTGACTTCCTTGAGGAACACATGATGCGTCCTAC junctionGGTTGCTGCTACGCATATCATTGAGATGTCTGTGGGAGGAGTTGATGTGTACTCTGAGGACGATGAGGGTTACGGTACGTCTTTCATTGAGTGGTGATTTATGCATTAGGACTGCATAGGGATGCACTATAGACCACGGATGGTCAGTTCTTTAAGTTACTGAAAAGACACGATAAATTAATACGACTCACTATAGGGAGAGGAGGGACGAAAGGTTACTATATAGATACTGAATGAATACTTATAGAGTGCATAAAGTATGCATAATGGTGTACCTAGAGTGACCTCTAAGAATGGTGATTATATTGTATTAGTATCACCTTAACTTAAGGACCAACATAAAGGGAGGAGACTCATGTTCCGCTTATTGTTGAACCTACTGCGGCATAGAGTCACCTACCGATTTCTTGTGGTACTTTGTGCTGCCCTTGGGTACGCATCTCTTACTGGAGACCTCAGTTCACTGGAGTCTGTCGTTTGCTCTATACTCACTTGTAGCGATTAGGGTCTTCCTGACCGACTGATGGCTCACCGAGGGATTCAGCGGTATGATTGCATCACACCACTTCATCCCTATAGAGTCAAGTCCTAAGGTATACCCATAAAGAGCCTCTAATGGTCTATCCTAAGGTCTATACCTAAAGATAGGCCATCCTATCAGTGTCACCTAAAGAGGGTCTTAGAGAGGGCCTATGGAGTTCCTATAGGGTCCTTTAAAATATACCATAAAAATCTGAGTGACTATCTCACAGTGTACGGACCTAAAGTTCCCCCATAGGGGGTACCTAAAGCCCAGCCAATCACCTAAAGTCAACCTTCGGTTGACCTTGAGGGTTCCCTAAGGGTTGGGGATGACCCTTGGGTTTGTCTTTGGGTGTTACCTTGAGTGTCTCTCTGTGTCCCTATCTGTTACAGTCTCCTAAAGTATCCTCCTAAAGTCACCTCCTAACGTCCATCCTAAAGCCAACACCTAAAGCCTACACCTAAAGACCCATCAAGTCAACGCCTATCTTAAAGTTTAAACA TAAAGACCAGAOrigin of Replication

The present invention envisions the use of origins of replication knownin the art that have been identified from species-specific plasmid DNAs(e.g. CoIE1, R1, pT181, pC194, pE194, RSF1010, pSC101, pMB1, R6K, RK2,p15a, pBBR1, pUC, pBR322 and the like), from bacterial virus (e.g.φX174, M13, F1 and P4) and from bacterial chromosomal origins ofreplication (e.g. oriC).

Bacteria-Specific Origins of Replication

Plasmid replication depends on host enzymes and on plasmid-controlledcis and trans determinants. For example, some plasmids have determinantsthat are recognized in almost all gram-negative bacteria and actcorrectly in each host during replication initiation and regulation.Other plasmids possess this ability only in some bacteria (Kues, U andStahl, U 1989 Microbiol Rev 53:491-516).

Plasmids are replicated by three general mechanisms, namely theta type,strand displacement, and rolling circle (reviewed by Del Solar et al.1998 Microbio and Molec Biol. Rev 62:434-464) that start at the originof replication. This replication origins contain sites that are requiredfor interactions of plasmid and/or host encoded proteins.

Origins of replication used on the payload of the invention may bemoderate copy number, such as colE1 ori from pBR322 (15-20 copies percell) or the R6K plasmid (15-20 copies per cell) or may be high copynumber, e.g. pUC oris (500-700 copies per cell), pGEM oris (300-400copies per cell), pTZ oris (>1000 copies per cell) or pBluescript oris(300-500 copies per cell).

In one embodiment, the bacterial origin of replication is selected inthe group consisting of ColE1, pMB1 and variants (pBR322, pET, pUC,etc.), p15a, ColA, ColE2, pOSAK, pSC101, R6K, IncW (pSa etc.), IncFII,pT181, P1, F IncP, IncC, IncJ, IncN, IncP1, IncP4, IncQ, IncH11,RSF1010, CloDF13, NTP16, R1, f5, pPS10, pC194, pE194, BBR1, pBC1, pEP2,pWVO1, pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, pIJ101, pSN22,pAMbeta1, pIP501, pIP407, ZM6100(Sa), pCU1, RA3, pMOL98,RK2/RP4/RP1/R68, pB10, R300B, pRO1614, pRO1600, pECB2, pCM1, pFA3,RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158and pUB113.

More preferably, the bacterial origin of replication is a E. coli originof replication selected in the group consisting of ColE1, pMB1 andvariants (pBR322, pET, pUC, etc.), p15a, ColA, ColE2, pOSAK, pSC101,R6K, IncW (pSa etc.), IncFII, pT181, P1, F IncP, IncC, IncJ, IncN,IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5, pPS10.

More preferably, the bacterial origin of replication is selected in thegroup consisting of pC194, pE194, BBR1, pBC1, pEP2, pWVO1, pLF1311,pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, 0.1101, pSN22, pAMbeta1,pIP501, pIP407, ZM6100(Sa), pCU1, RA3, pMOL98, RK2/RP4/RP1/R68, pB10,R300B, pRO1614, pRO1600, pECB2, pCM1, pFA3, RepFIA, RepFIB, RepFIC,pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.

Even more preferably, the bacterial origin of replication is ColE1.

Phage Origin of Replication

The payload according to the invention may comprise a phage replicationorigin which can initiate, with complementation of a complete phagegenome, the replication of the payload for later encapsulation into thedifferent bacterial delivery vehicles.

A phage origin of replication comprised in the payload of the inventioncan be any origin of replication found in a phage. Preferably, the phageorigin of replication can be the wild-type or non-wildtype sequence ofthe M13, f1, φX174, P4, Lambda, P2, Lambda-like, HK022, mEP237, HK97,HK629, HK630, mEP043, mEP213, mEP234, mEP390, mEP460, mEPx1, mEPx2,phi80, mEP234, T2, T4, T5, T7, RB49, phiX174, R17, PRD1 P1-like,P2-like, P22, P22-like, N15 and N15-like bacteriophages.

More preferably, the phage origin of replication is selected in thegroup consisting of phage origins of replication of M13, f1, φX174, P4,and Lambda.

In a particular embodiment, the phage origin of replication is the P4origin of replication.

Promoters

As known by the person skilled in the art, a promoter may be classifiedas strong or weak according to its affinity for RNA polymerase. Thestrength of a promoter may depend on whether initiation of transcriptionoccurs at that promoter with high or low frequency. Different promoterswith different strengths may be used in the present invention leading todifferent levels of gene/protein expression (e.g. the level ofexpression initiated from an mRNA originating from a weak promoter islower than the level of expression initiated from a strong promoter).

It will be appreciated by those of ordinary skill in the art that apromoter sequence may be selected from a large number of known bacterialgenes expressed by various bacterial species. Also, method ofprokaryotic promoter prediction exists, and can be based on DNAstability analysis as described in Kanhere and Bansal (BMCBioinformatics 2005, 6:1). The choice of promoter on the payloadaccording to the present invention can thus be made based on thebacteria to target.

In some embodiments, a nucleic acid sequence of interest may bepositioned under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with thenucleic acid sequence of interest in its natural environment.

Examples of bacterial promoters for use in accordance with the presentinvention include, without limitation, positively regulated E. colipromoters such as positively regulated σ 70 promoters (e.g., induciblepBad/araC promoter, Lux cassette right promoter, modified lambda Prmpromote, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad,P(Las) TetO, P(Las) CIO, P(Rh1), Pu, FecA, pRE, cadC, hns, pLas, pLux),a “s” promoter (e.g., Pdps), σ 32 promoters (e.g., heat shock) and σ 54promoters (e.g., glnAp2); negatively regulated E. coli promoters such asnegatively regulated σ 70 promoters (e.g., Promoter (PRM+), modifiedlambda Prm promoter, TetR-TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ,RecA_DlexO_DLac01, dapAp, FecA, Pspac-hy, pel, plux-cl, plux-lac, CinR,CinL, glucose controlled, modified Pr, modifed Prm+, FecA, Pcya, rec A(SOS), Rec A (SOS), EmrR_regulated, Betl_regulated, pLac_lux, pTet_Lac,pLac/Mnt, pTet/Mnt, LsrA/cI, pLux/cI, Lad, LacIQ, pLacIQ1, pLas/cI,pLas/Lux, pLux/Las, pRecA with LexA binding site, reverse BBa_R0011,pLacl/ara-1, pLacIq, rrnB PI, cadC, hns, PfhuA, pBad/araC, nhaA, OmpF,RcnR), σ S promoters (e.g., Lutz-Bujard LacO with alternative sigmafactor σ 38), σ 32 promoters (e.g., Lutz-Bujard LacO with alternativesigma factor σ 32), σ 54 promoters (e.g., glnAp2); negatively regulatedB. subtilis promoters such as repressible B. subtilis σ A promoters(e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank), σ promoters, andthe BioFAB promoters disclosed in Mutalik VK et al (Nature Methods,2013, 10: 354-360, see in particular the supplementary data) as well ason the BioFAB website (biofab.synberc.org). Other inducible microbialpromoters and/or bacterial promoters may be used in accordance with thepresent invention. An inducible promoter for use in accordance with thepresent disclosure may be induced by (or repressed by) one or morephysiological condition(s), such as changes in pH, temperature,radiation, osmotic pressure, saline gradients, cell surface binding, andthe concentration of one or more extrinsic or intrinsic inducingagent(s). The extrinsic inducer or inducing agent may comprise, withoutlimitation, amino acids and amino acid analogs, saccharides andpolysaccharides, nucleic acids, protein transcriptional activators andrepressors, cytokines, toxins, petroleum-based compounds, metalcontaining compounds, salts, ions, enzyme substrate analogs, hormones orcombinations thereof.

Particularly preferred bacterial promoters for use in accordance withthe present invention may be selected from constitutive promotersregulated by σ 70 such as the promoters of the Anderson collection(parts.igem.org): BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23103,BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23108, BBa_J23109,BBa_J23110, BBa_J23111, BBa_J23112, BBa_J23113, BBa_J23114, BBa_J23115,BBa_J23116, BBa_J23117, BBa_J23118, and BBa_J23119.

In some embodiments of the present invention, a promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence downstream of the promoter. The enhancer may belocated at any functional location before or after the promoter.

Terminators

In some embodiments, the payload may comprise a terminator sequence, orterminator. A “terminator,” as used herein, is a nucleic acid sequencethat causes transcription to stop. A terminator may be unidirectional orbidirectional. It is comprised of a DNA sequence involved in specifictermination of an RNA transcript by an RNA polymerase. A terminatorsequence prevents transcriptional activation of downstream nucleic acidsequences by upstream promoters. Thus, in certain embodiments, aterminator that ends the production of an RNA transcript iscontemplated. A terminator may be necessary in vivo to achieve desirablegene/protein expression levels.

The most commonly used type of terminator is a forward terminator. Whenplaced downstream of a nucleic acid sequence of interest that is usuallytranscribed, a forward transcriptional terminator will causetranscription to abort. In some embodiments, bidirectionaltranscriptional terminators are provided, which usually causetranscription to terminate on both the forward and reverse strand. Insome embodiments, reverse transcriptional terminators are provided,which usually terminate transcription on the reverse strand only. Inprokaryotic systems, terminators usually fall into two categories (1)rho-independent terminators and (2) rho-dependent terminators.Rho-independent terminators are generally composed of palindromicsequence that forms a stem loop rich in G-C base pairs followed by astring of uracil bases.

Terminators for use in accordance with the present invention include anyterminator of transcription described herein or known to one of ordinaryskill in the art. Examples of terminators include, without limitation,the termination sequences of genes such as, for example, the bovinegrowth hormone terminator, and viral termination sequences such as, forexample, the TO terminator, the TE terminator, Lambda T1 and the T1T2terminator found in bacterial systems. In some embodiments, thetermination signal may be a sequence that cannot be transcribed ortranslated, such as those resulting from a sequence truncation.

Terminators for use in accordance with the present invention alsoinclude terminators disclosed in Chen Y J et al (2013, Nature Methods,10: 659-664), and the BioFAB terminators disclosed in Cambray G et al(Nucl Acids Res, 2013, 41(9): 5139-5148).

Sequence of Interest Under the Control of the Promoter

According to the invention, the payload comprises a sequence of interestunder the control of a promoter.

In one embodiment, the sequence of interest is a programmable nucleasecircuits to be delivered to the targeted bacteria. This programmablenuclease circuit may be able to mediate in vivo sequence-specificelimination of bacteria that contain a target gene of interest (e.g. agene that is harmful to humans). Some embodiments of the presentdisclosure relate to engineered variants of the Type II CRISPR-Cas(Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated) system of Streptococcus pyogenes. Otherprogrammable nucleases that can be used include other CRISPR-Cassystems, engineered TALEN (Transcription Activator-Like EffectorNuclease) variants, engineered zinc finger nuclease (ZFN) variants,natural, evolved or engineered meganuclease or recombinase variants, andany combination or hybrids of programmable nucleases. Thus, theengineered autonomously distributed circuits provided herein may be usedto selectively cleave DNA encoding a gene of interest such as, forexample, a toxin gene, a virulence factor gene, an antibiotic resistancegene, a remodeling gene or a modulatory gene (cf. WO2014124226 andUS2015/0064138).

Other sequences of interest, preferably programmable, can be added tothe payload so as to be delivered to targeted bacteria. Preferably, thesequence of interest added to the payload leads to cell death of thetargeted bacteria. For example, the nucleic acid sequence of interestadded to the payload may encode holins or toxins.

Alternatively, the sequence of interest circuit added to the payloaddoes not lead to bacteria death. For example, the sequence of interestmay encode reporter genes leading to a luminescence or fluorescencesignal, or being able to elicit an immune response. Alternatively, thesequence of interest may comprise proteins and enzymes achieving auseful function such as modifying the metabolism of the bacteria or thecomposition of its environment and/or such as producing a therapeuticeffect.

In a particular embodiment, the nucleic sequence of interest is selectedin the group consisting of a Cas nuclease, a Cas9 nuclease, a guide RNA,a single guide RNA (sgRNA), a CRISPR locus, a gene expressing an enzymesuch as a nuclease or a kinase, a TALEN, a ZFN, a meganuclease, arecombinase, a bacterial receptor, a membrane protein, a structuralprotein, a secreted protein, resistance to an antibiotic or to a drug ingeneral, a gene expressing a toxic protein or a toxic factor and a geneexpressing a virulence protein or a virulence factor or any combinationthereof.

Toxin

In a particular embodiment, the payload according to the inventioncomprises a sequence of interest that encodes a bacteriocin, which canbe a proteinaceous toxin produced by bacteria to kill or inhibit growthof other bacteria. Bacteriocins are categorized in several ways,including producing strain, common resistance mechanisms, and mechanismof killing. Such bacteriocin had been described from gram negativebacteria (e.g. microcins, colicin-like bacteriocins and tailocins) andfrom gram positive bacteria (e.g. Class I, Class II, Class III or ClassIV bacteriocins).

In one embodiment, the payload according to the invention furthercomprises a sequence of interest encoding a toxin selected in the groupconsisting of microcins, colicin-like bacteriocins, tailocins, Class I,Class II, Class III and Class IV bacteriocins.

In a particular embodiment, the corresponding immunity polypeptide (i.e.anti-toxin) may be used to protect bacterial cells (see review by Cotteret al., Nature Reviews Microbiology 11: 95, 2013, which is herebyincorporated by reference in its entirety) for payload production andpackaging purpose but is absent in the pharmaceutical composition and inthe targeted bacteria in which the payload of the invention isdelivered.

CRISPR-Cas

The CRISPR system contains two distinct elements, i.e. i) anendonuclease, in this case the CRISPR associated nuclease (Cas or“CRISPR associated protein”) and ii) a guide RNA. The guide RNA is inthe form of a chimeric RNA which consists of the combination of a CRISPR(RNAcr) bacterial RNA and a RNAtracr (trans-activating RNA CRISPR)(Jinek et al., Science 2012). The gRNA combines the targetingspecificity of the cRNA corresponding to the “spacing sequences” thatserve as guides to the Cas proteins, and the conformational propertiesof the Rtracr in a single transcript. When the gRNA and the Cas proteinare expressed simultaneously in the cell, the target genomic sequencecan be permanently modified or interrupted. The modification isadvantageously guided by a repair matrix.

The CRISPR system includes two main classes depending on the nucleasemechanism of action:

-   -   Class 1 is made of multi-subunit effector complexes and includes        type I, III and IV    -   Class 2 is made of single-unit effector modules, like Cas9        nuclease, and includes type II (II-A,II-B,II-C,II-C variant), V        (V-A,V-B,V-C,V-D,V-E,V-U1,V-U2,V-U3,V-U4,V-U5) and VI        (VI-A,VI-B1,VI-B2,VI-C,VI-D)

The sequence of interest according to the present invention comprises anucleic acid sequence encoding Cas protein. A variety of CRISPR enzymesare available for use as a sequence of interest on the payload accordingto the present invention. In some embodiments, the CRISPR enzyme is aType II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzesDNA cleavage. In some other embodiments, the CRISPR enzyme catalyzes RNAcleavage. In one embodiment, the CRISPR enzymes may be coupled to asgRNA. In certain embodiments, the sgRNA targets a gene selected in thegroup consisting of an antibiotic resistance gene, virulence protein orfactor gene, toxin protein or factor gene, a bacterial receptor gene, amembrane protein gene, a structural protein gene, a secreted proteingene and a gene expressing resistance to a drug in general.

Non-limiting examples of Cas proteins as part of a multi-subuniteffector or as a single-unit effector include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Cas11 (SS), Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d(CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2),Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Csc1, Csc2, Cse1, Cse2,Csy1, Csy2, Csy3, Csf1, Csf2, Csf3, Csf4, Csm2, Csm3, Csm4, Csm5, Csm6,Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx13, Csx1, Csx15, SdCpf1, CmtCpf1, TsCpf1,CmaCpf1, PcCpf1, ErCpf1, FbCpf1, UbcCpf1, AsCpf1, LbCpf1, homologuesthereof, orthologues thereof, variants thereof, or modified versionsthereof. In some embodiments, the CRISPR enzyme cleaves both strands ofthe target nucleic acid at the Protospacer Adjacent Motif (PAM) site.

In a particular embodiment, the CRISPR enzyme is any Cas9 protein, forinstance any naturally-occurring bacterial Cas9 as well as any variants,homologs or orthologues thereof.

By “Cas9” is meant a protein Cas9 (also called Csnl or Csx12) or afunctional protein, peptide or polypeptide fragment thereof, i.e.capable of interacting with the guide RNA(s) and of exerting theenzymatic activity (nuclease) which allows it to perform thedouble-strand cleavage of the DNA of the target genome. “Cas9” can thusdenote a modified protein, for example truncated to remove domains ofthe protein that are not essential for the predefined functions of theprotein, in particular the domains that are not necessary forinteraction with the gRNA (s).

The sequence encoding Cas9 (the entire protein or a fragment thereof) asused in the context of the invention can be obtained from any known Cas9protein (Fonfara et al., 2014; Koonin et al., 2017). Examples of Cas9proteins useful in the present invention include, but are not limitedto, Cas9 proteins of Streptococcus pyogenes (SpCas9), Streptococcusthermophiles (St1Cas9, St3Cas9), Streptococcus mutans, Staphylococcusaureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida(FnCas9) and Neisseria meningitides (NmCas9).

The sequence encoding Cpf1 (Cas12a) (the entire protein or a fragmentthereof) as used in the context of the invention can be obtained fromany known Cpf1 (Cas12a) protein (Koonin et al., 2017). Examples of Cpf1(Cas12a) proteins useful in the present invention include, but are notlimited to, Cpf1(Cas12a) proteins of Acidaminococcus sp, Lachnospiraceaebacteriu and Francisella novicida.

The sequence encoding Cas13a (the entire protein or a fragment thereof)as used in the context of the invention can be obtained from any knownCas13a (C2c2) protein (Abudayyeh et al., 2017). Examples of Cas13a(C2c2) proteins useful in the present invention include, but are notlimited to, Cas13a (C2c2) proteins of Leptotrichia wadei (LwaCas13a).

The sequence encoding Cas13d (the entire protein or a fragment thereof)as used in the context of the invention can be obtained from any knownCas13d protein (Yan et al., 2018). Examples of Cas13d proteins useful inthe present invention include, but are not limited to, Cas13d proteinsof Eubacterium siraeum and Ruminococcus sp.

In a particular embodiment, the nucleic sequence of interest is aCRISPR/Cas9 system for the reduction of gene expression or inactivationa gene selected in the group consisting of an antibiotic resistancegene, virulence factor or protein gene, toxin factor or protein gene, agene expressing a bacterial receptor, a membrane protein, a structuralprotein, a secreted protein, and a gene expressing resistance to a drugin general.

In one embodiment, the CRISPR system is used to target and inactivate avirulence factor. A virulence factor can be any substance produced by apathogen that alter host-pathogen interaction by increasing the degreeof damage done to the host. Virulence factors are used by pathogens inmany ways, including, for example, in cell adhesion or colonization of aniche in the host, to evade the host's immune response, to facilitateentry to and egress from host cells, to obtain nutrition from the host,or to inhibit other physiological processes in the host. Virulencefactors can include enzymes, endotoxins, adhesion factors, motilityfactors, factors involved in complement evasion, and factors thatpromote biofilm formation. For example, such targeted virulence factorgene can be E. coli virulence factor gene such as, without limitation,EHEC-H1yA, Stx1 (VT1), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c(VT2c), Stx2d (VT2d), Stx2e (VT2e) and Stx2f (VT2f), Stx2h (VT2h), fimA,fimF, fimH, neuC, kpsE, sfa, foc, iroN, aer, iha, papC, papGI, papGII,papGIII, h1yC, cnf1, hra, sat, ireA, usp ompT, ibeA, malX, fyuA, irp2,traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aaiC, aatA, TEM, CTX,SHV, csgA, csgB, csgC, csgD, csgE, csgF, csgG, csgH, T1SS, T2SS, T3SS,T4SS, T5SS, T6SS (secretion systems). For example, such targetedvirulence factor gene can be Shigella dysenteriae virulence factor genesuch as, without limitation, stx1 and stx2. For example, such targetedvirulence factor gene can be Yersinia pestis virulence factor gene suchas, without limitation, yscF (plasmid-borne (pCD1) T3SS external needlesubunit). For example, such targeted virulence factor gene can beFrancisella tularensis virulence factor gene such as, withoutlimitation, fs1A. For example, such targeted virulence factor gene canbe Bacillus anthracia virulence factor gene such as, without limitation,pag (Anthrax toxin, cell-binding protective antigen). For example, suchtargeted virulence factor gene can be Vibrio cholera virulence factorgene such as, without limitation, ctxA and ctxB (cholera toxin), tcpA(toxin co-regulated pilus), and toxT (master virulence regulator). Forexample, such targeted virulence factor gene can be Pseudomonasaeruginosa virulence factor genes such as, without limitation,pyoverdine (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl,pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genespvdE, pvdR, pvdT, opmQ), siderophore pyochelin (e.g., pchD, pchC, pchB,pchA, pchE, pchF and pchG, and toxins (e.g., exoU, exoS and exoT). Forexample, such targeted virulence factor gene can be Klebsiellapneumoniae virulence factor genes such as, without limitation, fimA(adherence, type I fimbriae major subunit), and cps (capsularpolysaccharide). For example, such targeted virulence factor gene can beAcinetobacter baumannii virulence factor genes such as, withoutlimitation, ptk (capsule polymerization) and epsA (assembly). Forexample, such targeted virulence factor gene can be Salmonella entericaTyphi virulence factor genes such as, without limitation, MIA (invasion,SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with biletolerance, including efflux pump genes acrA, acrB and to 1C. Forexample, such targeted virulence factor gene can be Fusobacteriumnucleatum virulence factor genes such as, without limitation, FadA andTIGIT. For example, such targeted virulence factor gene can beBacteroides fragilis virulence factor genes such as, without limitation,bft.

In another embodiment, the CRISPR/Cas9 system is used to target andinactivate an antibiotic resistance gene such as, without limitation,GyrB, ParE, ParY, AAC(1), AAC(2′), AAC(3), AAC(6′), ANT(2″), ANT(3″),ANT(4′), ANT(6), ANT(9), APH(2″), APH(3″), APH(3′), APH(4), APH(6),APH(7″), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-M, KPC,SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXAβ-lactamase, mecA, Omp36, OmpF, PIB, bla (blal, blaR1) and mec (mecl,mecR1) operons, Chloramphenicol acetyltransferase (CAT), Chloramphenicolphosphotransferase, Ethambutol-resistant arabinosyltransferase (EmbB),MupA, MupB, Integral membrane protein MprF, Cfr 23 S rRNAmethyltransferase, Rifampin ADP-ribosyltransferase (Arr), Rifampinglycosyltransferase, Rifampin monooxygenase, Rifampinphosphotransferase, DnaA, RbpA, Rifampin-resistant beta-subunit of RNApolymerase (RpoB), Erm 23 S rRNA methyltransferases, Lsa, MsrA, Vga,VgaB, Streptogramin Vgb lyase, Vat acetyltransferase, Fluoroquinoloneacetyltransferase, Fluoroquinolone-resistant DNA topoisomerases,Fluoroquinolone-resistant GyrA, GyrB, ParC, Quinolone resistance protein(Qnr), FomA, FomB, FosC, FosA, FosB, FosX, VanA, VanB, VanD, VanR, VanS,Lincosamide nucleotidyltransferase (Lin), EreA, EreB, GimA, Mgt, Ole,Macrolide phosphotransferases (MPH), MefA, MefE, Mel, Streptothricinacetyltransferase (sat), Sul1, Sul2, Sul3, sulfonamide-resistant FolP,Tetracycline inactivation enzyme TetX, TetA, TetB, TetC, Tet30, Tet31,TetM, TetO, TetQ, Tet32, Tet36, MacAB-To1C, MsbA, MsrA, VgaB, EmrD,EmrAB-To1C, NorB, GepA, MepA, AdeABC, AcrD, MexAB-OprM, mtrCDE, EmrE,adeR, acrR, baeSR, mexR, phoPQ, mtrR, or any antibiotic resistance genedescribed in the Comprehensive Antibiotic Resistance Database(card.mcmaster.ca)

In another embodiment, the CRISPR/Cas9 system is used to target andinactivate a bacterial toxin gene. Bacterial toxin can be classified aseither exotoxins or endotoxins. Exotoxins are generated and activelysecreted; endotoxins remain part of the bacteria. The response to abacterial toxin can involve severe inflammation and can lead to sepsis.Such toxin can be for example Botulinum neurotoxin, Tetanus toxin,Staphylococcus toxins, Diphtheria toxin, Anthrax toxin, Alpha toxin,Pertussis toxin, Shiga toxin, Heat-stable enterotoxin (E. coli ST),colibactin, BFT (B. fragilis toxin) or any toxin described in Henkel etal., (Toxins from Bacteria in EXS. 2010; 100: 1-29).

Selection Marker

In one embodiment, the payload according to the invention may furthercomprise a selection marker. In a particular embodiment, the selectionmarker provides a selective advantage to the bacterial cell infected bythe payload, such as resistance to antibiotics, resistance to heavymetals, complementing a host auxotrophy and/or exhibiting fluorescent orluminescent proteins.

The inclusion of the suitable selectable marker gene in a payload allowstesting and/or detection for successful delivery of the payloadaccording to the invention. The payload according to the invention maycomprise one or more nucleic acid sequences encoding selectable markersuch as auxotrophic markers (e.g., LEU2, URA3, TRP 1, HIS3, DapA orThyA) (Peubez et al, 2010), detectable labels such as fluorescent orluminescent proteins (e.g., GFP, eGFP, DsRed, CFP, YFP), or proteinconferring resistance to a chemical/toxic compound (e.g., MGMT geneconferring resistance to temozolomide, kanamycin resistance,chloramphenicol resistance, etc.) or any combinations thereof. Thesemarkers can be used to select or detect host cells comprising the vectoraccording to the invention and can be easily chosen by the skilledperson according to the host cell.

For most purposes, an antibiotic resistance gene is a commonly usedselection marker to facilitate molecular biology cloning of the payloadand to allow the detection or selection of bacteria transformed by suchpayload. Antibiotic resistance genes are well known in the art andinclude but are not limited to ampicillin resistance (Amp),chloramphenicol resistance (Cm), tetracycline resistance (Tet),kanamycin resistance (Kan), hygromycin resistance (Qiyg or hph genes),and zeomycin resistance (Zeo).

Alternatively, antibiotic-free selection systems have been suggested.Such antibiotic-free selection systems include bacterial toxin-antitoxinsystems [Engelberg-Kulka, H. and Glaser, G., Annu. Rev. Microbiol. 53(1999) 43-70] and genes responsible for resistance against heavy metals,such as tellurium [Silver, S, and Phung, L. T., Annu. Rev. Microbiol. 50(1996) 753-789], and systems, in which the payload encodes a genecomplementing a host auxotrophy [Wang, M. D., et al., J. Bacteriol. 169(1987) 5610-5614].

Auxotrophic marker selection in bacteria has previously been described.See, for example, U.S. Pat. Nos. 4,920,048, 5,691,185, 6,291,245,6,413,768, 6,752,994, Struhl et al. ((1976) PNAS USA 73; 1471-1475);MacCormick, C. A., et al., ((1995) FEMS Microbiol. Lett. 127:105-109);Dickely et al. ((1995), Mol. Microbiol. 15:839-847); Sørensen et al.,((2000) Appl. Environ. Microbiol. 66:1253-1258); Fiedler & Skerra,((2001) Gene 274: 111-118).

In a preferred embodiment, the payload according to the inventioncomprises an auxotrophic marker.

Targeted Bacteria

The bacteria targeted by bacterial delivery vehicles can be any bacteriapresent in a mammal organism. It can be any commensal, symbiotic orpathogenic bacteria of the microbiota or microbiome.

A microbiome may comprise of a variety of endogenous bacterial species,any of which may be targeted in accordance with the present disclosure.In some embodiments, the genus and/or species of targeted endogenousbacterial cells may depend on the type of bacteriophages being used forpreparing the bacterial virus particles. For example, somebacteriophages exhibit tropism for, or preferentially target, specifichost species of bacteria. Other bacteriophages do not exhibit suchtropism and may be used to target a number of different genus and/orspecies of endogenous bacterial cells.

Examples of bacterial cells include, without limitation, cells frombacteria of the genus Yersinia, Escherichia, Klebsiella, Acinetobacter,Bordetella, Neisseria, Aeromonas, Franciesella, Corynebacterium,Citrobacter, Chlamydia, Hemophilus, Brucella, Mycobacterium, Legionella,Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus,Erysipelothrix, Salmonella, Streptomyces, Streptococcus, Staphylococcus,Bacteroides, Prevotella, Clostridium, Bifidobacterium, Clostridium,Brevibacterium, Lactococcus, Leuconostoc, Actinobacillus, Selnomonas,Shigella, Zymonas, Mycoplasma, Treponema, Leuconostoc, Corynebacterium,Enterococcus, Enterobacter, Pyrococcus, Serratia, Morganella,Parvimonas, Fusobacterium, Actinomyces, Porphyromonas, Micrococcus,Bartonella, Borrelia, Brucelia, Campylobacter, Chlamydophilia,Cutibacterium, Propionibacterium, Gardnerella, Ehrlichia, Haemophilus,Leptospira, Listeria, Mycoplasma, Nocardia, Rickettsia, Ureaplasma, andLactobacillus, and any mixture thereof.

Thus, delivery vehicles may target (e.g., specifically target) abacterial cell from any one or more of the foregoing genus of bacteriato specifically deliver the payload according to the invention.

Preferably, the targeted bacteria can be selected from the groupconsisting of Yersinia spp., Escherichia spp., Klebsiella spp.,Acinetobacter spp., Pseudomonas spp., Helicobacter spp., Vibrio spp.,Salmonella spp., Streptococcus spp., Staphylococcus spp., Bacteroidesspp., Clostridium spp., Shigella spp., Enterococcus spp., Enterobacterspp., Listeria spp., Cutibacterium spp., Propionibacterium spp.,Fusobacterium spp., Porphyromonas spp. and Gardnerella spp.

In some embodiments, bacterial cells of the present invention areanaerobic bacterial cells (e.g., cells that do not require oxygen forgrowth). Anaerobic bacterial cells include facultative anaerobic cellssuch as but not limited to Escherichia coli, Shewanella oneidensis,Gardnerella vaginalis and Listeria. Anaerobic bacterial cells alsoinclude obligate anaerobic cells such as, for example, Bacteroides,Clostridium, Cutibacterium, Propionibacterium, Fusobacterium andPorphyromonas species. In humans, anaerobic bacteria are most commonlyfound in the gastrointestinal tract. In some particular embodiment, thetargeted bacteria are thus bacteria most commonly found in thegastrointestinal tract. Bacteriophages used for preparing the bacterialvirus particles, and then the bacterial virus particles, may target(e.g., to specifically target) anaerobic bacterial cells according totheir specific spectra known by the person skilled in the art tospecifically deliver the payload.

In some embodiments, the targeted bacterial cells are, withoutlimitation, Bacteroides thetaiotaomicron, Bacteroides fragilis,Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum,Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis,Clostridium butyricum, Brevibacterium lactofermentum, Streptococcusagalactiae, Lactococcus lactis, Leuconostoc lactis, Actinobacillusactinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacterpylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis,Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis,Staphilococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis,Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei,Lactobacillus acidophilus, Enterococcus faecalis, Bacillus coagulans,Bacillus cereus, Bacillus popillae, Synechocystis strain PCC6803,Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium,Lactobacillus hilgardii, Streptococcus fetus, Lactobacillus pentosus,Bacteroides fragilis, Staphylococcus epidermidis, Streptomycesphaechromogenes, Streptomyces ghanaenis, Klebsiella pneumoniae,Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens,Morganella morganii, Citrobacter freundii, Propionibacteriumfreudenreichii, Pseudomonas aerigunosa, Parvimonas micra, Prevotellaintermedia, Fusobacterium nucleatum, Prevotella nigrescens, Actinomycesisraelii, Porphyromonas endodontalis, Porphyromonas gingivalisMicrococcus luteus, Bacillus megaterium, Aeromonas hydrophila, Aeromonascaviae, Bacillus anthracia, Bartonella henselae, Bartonella Quintana,Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borreliaafzelii, Borrelia recurrentis, Brucella abortus, Brucella canis,Brucella melitensis, Brucella suis, Campylobacter jejuni, Campylobactercoli, Campylobacter fetus, Chlamydia pneumoniae, Chlamydia trachomatis,Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria,Cutibacterium acnes (formerly Propionibacterium acnes), Ehrlichia canis,Ehrlichia chaffeensis, Enterococcus faecium, Francisella tularensis,Haemophilus influenza, Legionella pneumophila, Leptospira interrogans,Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeriamonocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae,Neisseria meningitides, Nocardia asteroids, Rickettsia rickettsia,Salmonella enteritidis, Salmonella typhi, Salmonella paratyphi,Salmonella typhimurium, Shigella flexnerii, Shigella dysenteriae,Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcuspyogenes, Gardnerella vaginalis, Streptococcus viridans, Treponemapallidum, Ureaplasma urealyticum, Vibrio cholera, Vibrioparahaemolyticus, Yersinia pestis, Yersinia enterocolitica, Yersiniapseudotuberculosis, Actinobacter baumanii, Pseudomonas aerigunosa, and amixture thereof, preferably the bacteria of interest are selected fromthe group consisting of Escherichia coli, Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii,Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacteraerogenes, and a mixture thereof.

In one embodiment, the targeted bacteria are Escherichia coli.

Thus, bacteriophages used for preparing the bacterial virus particles,and then the bacterial virus particles, may target (e.g., specificallytarget) a bacterial cell from any one or more of the foregoing genusand/or species of bacteria to specifically deliver the payload.

In one embodiment, the targeted bacteria are pathogenic bacteria. Thetargeted bacteria can be virulent bacteria.

The targeted bacteria can be antibacterial resistance bacteria,preferably selected from the group consisting of extended-spectrumbeta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiellapneumoniae, vancomycin-resistant Enterococcus (VRE),methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant(MDR) Acinetobacter baumannii, MDR Enterobacter spp., and a combinationthereof. Preferably, the targeted bacteria can be selected from thegroup consisting of extended-spectrum beta-lactamase-producing (ESBL)Escherichia coli strains.

Alternatively, the targeted bacterium can be a bacterium of themicrobiome of a given species, preferably a bacterium of the humanmicrobiota.

Bacterial Viruses

The bacterial virus particles are prepared from bacterial virus. Thebacterial viruses are chosen in order to be able to introduce thepayload into the targeted bacteria.

Bacterial viruses are preferably bacteriophages. Bacteriophage areobligate intracellular parasites that multiply inside bacteria byco-opting some or all of the host biosynthetic machinery. Phage genomescome in a variety of sizes and shapes (e.g., linear or circular). Mostphages range in size from 24-200 nm in diameter. Phages contain nucleicacid (i.e., genome) and proteins, and may be enveloped by a lipidmembrane. Depending upon the phage, the nucleic acid genome can beeither DNA or RNA, and can exist in either circular or linear forms. Thesize of the phage genome varies depending upon the phage. The simplestphages have genomes that are only a few thousand nucleotides in size,while the more complex phages may contain more than 100,000 nucleotidesin their genome, and in rare instances more than 1,000,000. The numberand amount of individual types of protein in phage particles will varydepending upon the phage.

Optionally, the bacteriophage is selected from the Order Caudoviralesconsisting of, based on the taxonomy of Krupovic et al, Arch Virol,2015:

-   -   family Myoviridae such as, without limitation, genus Cp220virus,        Cp8virus, Ea214virus, Felixo1virus, Mooglevirus, Suspvirus,        Hp1virus, P2virus, Kayvirus, P100virus, Silviavirus, Spo1virus,        Tsarbombavirus, Twortvirus, Cc31virus, Jd18virus, Js98virus,        Kp15virus, Moonvirus, Rb49virus, Rb69virus, S16virus,        Schizot4virus, Sp18virus, T4virus, Cr3virus, Se1virus, V5virus,        Abouovirus, Agatevirus, Agrican357virus, Ap22virus, Arv1virus,        B4virus, Bastillevirus, Bc431virus, Bcep78virus, Bcepmuvirus,        Biquartavirus, Bxz1virus, Cd119virus, Cp51virus, Cvm10virus,        Eah2virus, Elvirus, Hapunavirus, Jimmervirus, Kpp10virus,        M12virus, Machinavirus, Marthavirus, Msw3virus, Muvirus,        Myohalovirus, Nit1virus, P1virus, Pakpunavirus, Pbunavirus,        Phikzvirus, Rheph4virus, Rs12virus, Rs1unavirus, Secunda5virus,        Sep1virus, Spn3virus, Svunavirus, Tg1virus, Vhm1virus and        Wphvirus.    -   family Podoviridae such as, without limitation, genus Fri1virus,        Kp32virus, Kp34virus, Phikmvvirus, Pradovirus, Sp6virus,        T7virus, Cp1virus, P68virus, Phi29virus, Nona33virus, Pocjvirus,        Tl2011virus, Bcep22virus, Bpp1virus, Cba41virus, Dfl12virus,        Ea92virus, Epsilon15virus, F116virus, G7cvirus, Jwa1phavirus,        Kf1virus, Kpp25virus, Lit1virus, Luz24virus, Luz7virus, N4virus,        Nonanavirus, P22virus, Pagevirus, Phieco32virus, Prtbvirus,        Sp58virus, Una961virus and Vp5virus.    -   family Siphoviridae such as, without limitation, genus Camvirus,        Likavirus, R4virus, Acadianvirus, Coopervirus, Pg1virus,        Pipefishvirus, Rosebushvirus, Brujitavirus, Che9cvirus,        Hawkeyevirus, Plotvirus, Jerseyvirus, Klgvirus, Sp311virus,        Lmdlvirus, Una4virus, Bongovirus, Reyvirus, Buttersvirus,        Charlievirus, Redivirus, Baxtervirus, Nymphadoravirus,        Bignuzvirus, Fishburnevirus, Phayoncevirus, Kp36virus,        Rogue1virus, Rtpvirus, T1virus, T1svirus, Ab18virus, Amigovirus,        Anatolevirus, Andromedavirus, Attisvirus, Barnyardvirus,        Berna113virus, Biseptimavirus, Bronvirus, C2virus, C5virus,        Cba181virus, Cbastvirus, Cecivirus, Che8virus, Chivirus,        Cjw1virus, Corndogvirus, Cronusvirus, D3112virus, D3virus,        Decurrovirus, Demosthenesvirus, Doucettevirus, E125virus,        Eiauvirus, Ff47virus, Gaiavirus, Gilesvirus, Gordonvirus,        Gordtnkvirus, Harrisonvirus, Hk578virus, Hk97virus, Jenstvirus,        Jwxvirus, Kelleziovirus, Korravirus, L5virus, Lambdavirus,        Laroyevirus, Liefievirus, Marvinvirus, Mudcatvirus, N15virus,        Nonagvirus, Nplvirus, Omegavirus, P12002virus, P12024virus,        P23virus, P70virus, Pa6virus, Pamx74virus, Patiencevirus,        Pbilvirus, Pepy6virus, Pfr1virus, Phic31virus, Phicbkvirus,        Phietavirus, Phifelvirus, Phijllvirus, Pis4avirus, Psavirus,        Psimunavirus, Rdjlvirus, Rer2virus, Sap6virus, Send513virus,        Septima3virus, Seuratvirus, Sextaecvirus, Sfi11virus,        Sfi21dtivirus, Sitaravirus, Sk1virus, Slashvirus, Smoothievirus,        Soupsvirus, Spbetavirus, Ssp2virus, T5virus, Tankvirus,        Tin2virus, Titanvirus, Tm4virus, Tp21virus, Tp84virus,        Triavirus, Trigintaduovirus, Vegasvirus, Vendettavirus,        Wbetavirus, Wildcatvirus, Wizardvirus, Woesvirus, Xp10virus,        Ydn12virus and Yuavirus.    -   family Ackermannviridae such as, without limitation, genus        Ag3virus, Limestonevirus, Cba120virus and Vilvirus.

Optionally, the bacteriophage is not part of the Order Caudovirales butfrom families with Unassigned order such as, without limitation, familyTectiviridae (such as genus Alphatectivirus, Betatectivirus), familyCorticoviridae (such as genus Corticovirus), family Inoviridae (such asgenus Fibrovirus, Habenivirus, Inovirus, Lineavirus, Plectrovirus,Saetivirus, Vespertiliovirus), family Cystoviridae (such as genusCystovirus), family Leviviridae (such as genus Allolevivirus,Levivirus), family Microviridae (such as genus Alpha3microvirus,G4microvirus, Phix174microvirus, Bdellomicrovirus, Chlamydiamicrovirus,Spiromicrovirus) and family Plasmaviridae (such as genus Plasmavirus).

Optionally, the bacteriophage is targeting Archea not part of the OrderCaudovirales but from families with Unassigned order such as, withoutlimitation, Ampullaviridae, FuselloViridae, Globuloviridae,Guttaviridae, Lipothrixviridae, Pleolipoviridae, Rudiviridae,Salterprovirus and Bicaudaviridae.

A non-exhaustive listing of bacterial genera and their knownhost-specific bacteria viruses is presented in the following paragraphs.Synonyms and spelling variants are indicated in parentheses. Homonymsare repeated as often as they occur (e.g., D, D, d) Unnamed phages areindicated by “NN” beside their genus and their numbers are given inparentheses.

Bacteria of the genus Actinomyces can be infected by the followingphages: Av-I, Av-2, Av-3, BF307, CT1, CT2, CT3, CT4, CT6, CT7, CT8 and1281.

Bacteria of the genus Aeromonas can be infected by the following phages:AA-I, Aeh2, N, PM1, TP446, 3, 4, 11, 13, 29, 31, 32, 37, 43, 43-10T, 51,54, 55R.1, 56, 56RR2, 57, 58, 59.1, 60, 63, Aeh1, F, PM2, 1, 25, 31,40RR2.8t, (syn=44R), (syn=44RR2.8t), 65, PM3, PM4, PM5 and PM6.

Bacteria of the genus Bacillus can be infected by the following phages:A, aiz1, A1-K-I, B, BCJA1, BC1, BC2, BLL1, BL1, BP142, BSL1, BSL2, BS1,BS3, BS8, BS15, BS18, BS22, BS26, BS28, BS31, BS104, BS105, BS106, BTB,B1715V1, C, CK-I, Coll, Corl, CP-53, CS-I, CSi, D, D, D, D5, ent1, FPB,FP9, FSi, FS2, FS3, FS5, FS8, FS9, G, GH8, GT8, GV-I, GV-2, GT-4, g3,g12, g13, g14, g16, g17, g21, g23, g24, g29, H2, ken1, KK-88, Kum1,Kyu1, J7W-1, LP52, (syn=LP-52), L7, Mex1, MJ-I, mor2, MP-7, MP1O, MP12,MP14, MP15, Neo1, No 2, N5, N6P, PBC1, PBLA, PBP1, P2, S-a, SF2, SF6,Sha1, Si11, SP02, (syn=ΦSPP1), SPβ, STI, STi, SU-I1, t, TbI, Tb2, Tb5,TbIO, Tb26, Tb51, Tb53, Tb55, Tb77, Tb97, Tb99, Tb560, Tb595, Td8, Td6,Td15, TgI, Tg4, Tg6, Tg7, Tg9, TgIO, TgI1, Tg13, Tg15, Tg21, Tin1, Tin7,Ting, Tin13, Tm3, Toc1, Tog1, to11, TP-I, TP-10vir, TP-15c, TP-16c,TP-17c, TP-19, TP35, TP51, TP-84, Tt4, Tt6, type A, type B, type C, typeD, type E, Tφ3, VA-9, W, wx23, wx26, Yun1, α, γ, p11, φmed-2, φT, φμ-4,φ3T, φ75, φ1O5, (syn=φ1O5), IA, IB, 1-97A, 1-97B, 2, 2, 3, 3, 3, 5, 12,14, 20, 30, 35, 36, 37, 38, 41C, 51, 63, 64, 138D, I, II, IV,NN-Bacillus (13), ale1, AR1, AR2, AR3, AR7, AR9, Bace-11, (syn=11),Bastille, BL1, BL2, BL3, BL4, BL5, BL6, BL8, BL9, BP124, BS28, BS80, Ch,CP-51, CP-54, D-5, dar1, den1, DP-7, ent1, FoSi, FoS2, FS4, FS6, FS7, G,ga11, gamma, GE1, GF-2, GSi, GT-I, GT-2, GT-3, GT-4, GT-5, GT-6, GT-7,GV-6, g15, 19, 110, ISi, K, MP9, MP13, MP21, MP23, MP24, MP28, MP29,MP30, MP32, MP34, MP36, MP37, MP39, MP40, MP41, MP43, MP44, MP45, MP47,MP50, NLP-I, No. 1, N17, N19, PBS1, PK1, PMB1, PMB12, PMJ1, S, SPO1,SP3, SP5, SP6, SP7, SP8, SP9, SP1O, SP-15, SP50, (syn=SP-50), SP82, SST,subl, SW, Tg8, Tg12, Tg13, Tg14, thu1, thuΛ, thuS, Tin4, Tin23, TP-13,TP33, TP50, TSP-I, type V, type VI, V, Vx, β22, φe, φNR2, φ25, φ63, 1,1, 2, 2C, 3NT, 4, 5, 6, 7, 8, 9, 10, 12, 12, 17, 18, 19, 21, 138, III, 4(B. megateriwn), 4 (B. sphaericus), AR13, BPP-IO, BS32, BS107, B1, B2,GA-I, GP-IO, GV-3, GV-5, g8, MP20, MP27, MP49, Nf, PP5, PP6, SF5, Tg18,TP-I, Versailles, φ15, φ29, 1-97, 837/IV, m{umlaut over (ι)}-Bacillus(1), Bat1O, BSL1O, BSLI 1, BS6, BSI 1, BS16, BS23, BS1O1, BS102, g18,mor1, PBL1, SN45, thu2, thu3, TmI, Tm2, TP-20, TP21, TP52, type F, typeG, type IV, HN-BacMus (3), BLE, (syn=θc), BS2, BS4, BSS, BS7, B1O, B12,BS20, BS21, F, MJ-4, PBA12, AP50, AP50-04, AP50-11, AP50-23, AP50-26,AP50-27 and Bam35. The following Bacillus-specific phages are defective:DLP10716, DLP-11946, DPB5, DPB12, DPB21, DPB22, DPB23, GA-2, M, No. IM,PBLB, PBSH, PBSV, PBSW, PBSX, PBSY, PBSZ, phi, SPa, type 1 and μ.

Bacteria of the genus Bacteroides can be infected by the followingphages: ad I2, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, F1, β1, φA1,φBrO1, φBrO2, 11, 67.1, 67.3, 68.1, mt-Bacteroides (3), Bf42, Bf71,HN-Bdellovibrio (1) and BF-41.

Bacteria of the genus Bordetella can be infected by the followingphages: 134 and NN-Bordetella (3).

Bacteria of the genus Borrellia can be infected by the following phages:NN-Borrelia (1) and NN-Borrelia (2).

Bacteria of the genus Brucella can be infected by the following phages:A422, Bk, (syn=Berkeley), BM29, FOi, (syn=F01), (syn=FQ1), D, FP2,(syn=FP2), (syn=FD2), Fz, (syn=Fz75/13), (syn=Firenze 75/13), (syn=Fi),Fi, (syn=F1), Fim, (syn=FIm), (syn=Fim), FiU, (syn=HU), (syn=FiU), F2,(syn=F2), F3, (syn=F3), F4, (syn=F4), F5, (syn=F5), F6, F7, (syn=F7),F25, (syn=F25), (syn=£25), F25U, (syn=F25u), (syn=F25U), (syn=F25V),F44, (syn-F44), F45, (syn=F45), F48, (syn=F48), I, Im, M, MC/75, M51,(syn=M85), P, (syn=D), 5708, R, Tb, (syn=TB), (syn=Tbilisi), W,(syn=Wb), (syn=Weybridge), X, 3, 6, 7, 10/1, (syn=10), (syn=F8),(syn=F8), 12m, 24/11, (syn=24), (syn=F9), (syn=F9), 45/111, (syn=45),75, 84, 212/XV, (syn=212), (syn=Fi0), (syn=F10), 371/XXIX, (syn=371),(syn=Fn), (syn=F11) and 513.

Bacteria of the genus Burkholderia can be infected by the followingphages: CP75, NN-Burkholderia (1) and 42.

Bacteria of the genus Campylobacter can be infected by the followingphages: C type, NTCC12669, NTCC12670, NTCC12671, NTCC12672, NTCC12673,NTCC12674, NTCC12675, NTCC12676, NTCC12677, NTCC12678, NTCC12679,NTCC12680, NTCC12681, NTCC12682, NTCC12683, NTCC12684, 32f, 111c, 191,NN-Campylobacter (2), Vfi-6, (syn=V19), VfV-3, V2, V3, V8, V16,(syn=Vfi-1), V19, V20(V45), V45, (syn=V-45) and NN-Campylobacter (1).

Bacteria of the genus Chlamydia can be infected by the following phage:Chp1.

Bacteria of the genus Clostridium can be infected by the followingphages: CAK1, CA5, Ca7, CEβ, (syn=1C), CEγ, Cld1, c-n71, c-203 Tox-,DEβ, (syn=ID), (syn=1Dt0X+), HM3, KM1, KT, Ms, NA1, (syn=Naltox+),PA135Oe, Pfó, PL73, PL78, PL81, P1, P50, P5771, P19402, 1Ct0X+,2Ct0X\2D3 (syn=2Dt0X+), 3C, (syn=3Ctox+), 4C, (syn=4Ct0X+), 56, III-1,NN-Clostridium (61), NB1t0X+, α1, CA1, HMT, HM2, PF15 P-23, P-46, Q-05,Q-oe, Q-16, Q-21, Q-26, Q-40, Q-46, S111, SA02, WA01, WA03, Wm, W523,80, C, CA2, CA3, CPT1, CPT4, c1, c4, c5, HM7, H11/A1, H18/Ax, FWS23,Hi58ZA1, K2ZA1, K21ZS23, ML, NA2t0X; Pf2, Pf3, Pf4, S9ZS3, S41ZA1,S44ZS23, α2, 41, 112ZS23, 214/S23, 233/Ai, 234/S23, 235/S23, II-1, 11-2,11-3, NN-Clostridium (12), CA1, F1, K, S2, 1, 5 and NN-Clostridium (8).

Bacteria of the genus Corynebacterium can be infected by the followingphages: CGK1 (defective), A, A2, A3, A101, A128, A133, A137, A139, A155,A182, B, BF, B17, B18, B51, B271, B275, B276, B277, B279, B282, C, capi,CC1, CG1, CG2, CG33, CL31, Cog, (syn=CG5), D, E, F, H, H-I, hqi, hq2,11ZH33, Ii/31, J, K, K, (syn=Ktox”), L, L, (syn=Ltox+), M, MC-I, MC-2,MC-3, MC-4, MLMa, N, O, ovi, ov2, ov3, P, P, R, RP6, RS29, S, T, U, UB1,ub2, UH1, UH3, uh3, uh5, uh6, β, (syn=βtox+), βhv64, βvir, γ,(syn=γtoχ−), γ19, δ, (syn=δ′ox+), p, (syn=ptoχ−), Φ9, φ84, ω, IA,1/1180, 2, 2/1180, 5/1180, 5ad/9717, 7/4465, 8/4465, 8ad/10269, 10/9253,13Z9253, 15/3148, 21/9253, 28, 29, 55, 2747, 2893, 4498 and 5848.

Bacteria of the genus Enterococcus are infected by the following phage:DF78, F1, F2, 1, 2, 4, 14, 41, 867, D1, SB24, 2BV, 182, 225, C2, C2F,E3, E62, DS96, H24, M35, P3, P9, SB1O1, S2, 2B II, 5, 182a, 705, 873,881, 940, 1051, 1057, 21096C, NN-Enterococcus (1), PE1, F1, F3, F4,VD13, 1, 200, 235 and 341.

Bacteria of the genus Erysipelothrix can be infected by the followingphage: NN-Eiysipelothrix (1).

Bacteria of the genus Escherichia can be infected by the followingphages: BW73, B278, D6, D108, E, E1, E24, E41, FI-2, FI-4, FI-5, HI8A,Ff18B, i, MM, Mu, (syn=mu), (syn=MuI), (syn=Mu-I), (syn=MU-I),(syn=MuI), (syn=μ), 025, PhI-5, Pk, PSP3, P1, P1D, P2, P4 (defective),S1, Wφ, φK13, φR73 (defective), φ1, φ2 , φ7, φ92, iv (defective), 7 A,8φ, 9φ, 15 (defective), 18, 28-1, 186, 299, HH-Escherichia (2), AB48,CM, C4, C16, DD-VI, (syn=Dd-Vi), (syn=DDVI), (syn=DDVi), E4, E7, E28,FI1, FI3, H, H1, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7,Ox-I (syn=OX1), (syn=HF), Ox-2 (syn=0x2), (syn=0X2), Ox-3, Ox-4, Ox-5,(syn=0X5), Ox-6, (syn=66F), (syn=φ66t), (syn=φ66t−)5 0111, PhI-I, RB42,RB43, RB49, RB69, S, SaI-I, Sa1-2, Sa1-3, Sa1-4, Sa1-5, Sa1-6, TC23,TC45, TuII*-6, (syn=TuII*), TuIP-24, TuII*46, TuIP-60, T2,(syn=ganuTia), (syn=γ), (syn=PC), (syn=P.C.), (syn=T-2), (syn=T2),(syn=P4), T4, (syn=T-4), (syn=T4), T6, T35, α1, 1, IA, 3, (syn=Ac3), 3A,3T+, (syn=3), (syn=M1), 5φ, (syn=φ5), 9266Q, CFO103, HK620, J, K, K1F,m59, no. A, no. E, no. 3, no. 9, N4, sd, (syn=Sd), (syn=SD), (syn=Sa)3(syn=sd), (syn=SD), (syn=CD), T3, (syn=T-3), (syn=T3), T7, (syn=T-7),(syn=T7), WPK, W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, Φ04-CF, Φ05, Φ06,Φ07, φ1, φ1.2, φ20, φ95, φ263, φ1O92, φ1, φ11, (syn=φW), Ω8, 1, 3, 7, 8,26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W, NN-Escherichia (1),Esc-7-11, AC30, CVX-5, C1, DDUP, EC1, EC2, E21, E29, F1, F26S, F27S, Hi,HK022, HK97, (syn=ΦHK97), HK139, HK253, HK256, K7, ND-I, no.D, PA-2, q,S2, T1, (syn=α), (syn=P28), (syn=T-I), (syn=Tx), T3C, T5, (syn=T-5),(syn=T5), UC-I, w, β4, γ2, λ (syn=lambda), (syn=Φλ), ΦD326, φγ, Φ06, Φ7,Φ10, λ80, χ, (syn=χi), (syn=φχ), (syn=φχi), 2, 4, 4A, 6, 8A, 102, 150,168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243,K1O, ZG/3A, 5, 5A, 21EL, H19-J and 933H.

Bacteria of the genus Fusobacterium are infected by the following phage:NN-Fusobacterium (2), fv83-554/3, fv88-531/2, 227, fv2377, fv2527 andfv8501.

Bacteria of the genus Haemophilus are infected by the following phage:HP1, S2 and N3.

Bacteria of the genus Helicobacter are infected by the following phage:HP1 and {circumflex over ( )}{circumflex over ( )}-Helicobacter (1).

Bacteria of the genus Klebsiella are infected by the following phage:AIO-2, KI4B, K16B, K19, (syn=K19), K114, K115, K121, K128, K129, KI32,K133, K135, K1106B, K1171B, K1181B, K1832B, AIO-I, AO-I, AO-2, AO-3,FC3-10, K, K11, (syn=KI1), K12, (syn=K12), K13, (syn=K13), (syn=K170/11), K14, (syn=K14), K15, (syn=K15), K16, (syn=K16), K17, (syn=K17),K18, (syn=K18), K119, (syn=K19), K127, (syn=K127), K131, (syn=K131),K135, K1171B, II, VI, IX, CI-I, K14B, K18, K111, K112, K113, K116, K117,K118, K120, K122, K123, K124, K126, K130, K134, K1106B, KIi65B, K1328B,KLXI, K328, P5046, 11, 380, III, IV, VII, VIII, FC3-11, K12B,(syn=K12B), K125, (syn=K125), K142B, (syn=K142), (syn=K142B), K1181B,(syn=KI1 81), (syn=K1181B), K1765/!, (syn=K1765/1), K1842B,(syn=K1832B), K1937B, (syn=K1937B), L1, φ28, 7, 231, 483, 490, 632 and864/100.

Bacteria of the genus Lepitospira are infected by the following phage:LE1, LE3, LE4 and ˜NN-Leptospira (1).

Bacteria of the genus Listeria are infected by the following phage:A511, 01761, 4211, 4286, (syn=B054), A005, A006, A020, A500, A502, A511,A1 18, A620, A640, B012, B021, B024, B025, B035, B051, B053, B054, B055,B056, B101, BI 10, B545, B604, B653, C707, D441, H5047, H1OG, H8/73,H19, H21, H43, H46, H107, H108, HI 10, H163/84, H312, H340, H387,H391/73, H684/74, H924A, PSA, U153, φMLUP5, (syn=P35), 00241, 00611,02971A, 02971C, 5/476, 5/911, 5/939, 5/11302, 5/11605, 5/11704, 184,575, 633, 699/694, 744, 900, 1090, 1317, 1444, 1652, 1806, 1807,1921/959, 1921/11367, 1921/11500, 1921/11566, 1921/12460, 1921/12582,1967, 2389, 2425, 2671, 2685, 3274, 3550, 3551, 3552, 4276, 4277, 4292,4477, 5337, 5348/11363, 5348/11646, 5348/12430, 5348/12434, 10072,11355C, 11711A, 12029, 12981, 13441, 90666, 90816, 93253, 907515, 910716and NN-Lisferia (15).

Bacteria of the genus Morganella are infected by the following phage:47.

Bacteria of the genus Mycobacterium are infected by the following phage:13, AG1, ALi, ATCC 11759, A2, B.C3, BG2, BK1, BK5, butyricum, B-I, B5,B7, B30, B35, Clark, C1, C2, DNAIII, DSP1, D4, D29, GS4E, (syn=GS4E),GS7, (syn=GS-7), (syn=GS7), lacticola, Legendre, Leo, L5, (syn=ΦL-5),MC-I, MC-3, MC-4, minetti, MTPHI 1, Mx4, MyF3P/59a, ph1ei, (syn=ph1ei1), phlei 4, Polonus II, rabinovitschi, smegmatis, TM4, TM9, TM1O, TM20,Y7, Y1O, φ630, IB, IF, IH, 1/1, 67, 106, 1430, B1, (syn=Bo1), B24, D,D29, F-K, F-S, HP, Polonus I, Roy, R1, (syn=R1-Myb), (syn=Ri), 11, 31,40, 50, 103a, 103b, 128, 3111-D, 3215-D and NN-Mycobacterium (1).

Bacteria of the genus Neisseria are infected by the following phage:Group I, group II and NP1.

Bacteria of the genus Nocardia are infected by the following phage:MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.

Bacteria of the genus Proteus are infected by the following phage: Pm5,13vir, 2/44, 4/545, 6/1004, 13/807, 20/826, 57, 67b, 78, 107/69, 121,9/0, 22/608, 30/680, PmI, Pm3, Pm4, Pm6, Pm7, Pm9, PmIO, PmI1, Pv2, π1,φm, 7/549, 9B/2, 10A/31, 12/55, 14, 15, 16/789, 17/971, 19A/653, 23/532,25/909, 26/219, 27/953, 32A/909, 33/971, 34/13, 65, 5006M, 7480b, VI,13/3a, Clichy 12, π2600, φχ7, 1/1004, 5/742, 9, 12, 14, 22, 24/860,2600/D52, Pm8 and 24/2514.

Bacteria of the genus Providencia are infected by the following phage:PL25, PL26, PL37, 9211/9295, 9213/921 Ib, 9248, 7/R49, 7476/322,7478/325, 7479, 7480, 9000/9402 and 9213/921 Ia.

Bacteria of the genus Pseudomonas are infected by the following phage:PfI, (syn=Pf-I), Pf2, Pf3, PP7, PRR1, 7s, im-Pseudomonas (1), AI-I,AI-2, B 17, B89, CB3, Co1 2, Co1 11, Co1 18, Co1 21, C154, C163, C167,C2121, E79, F8, ga, gb, H22, K1, M4, N2, Nu, PB-I, (syn=PB1), pf16,PMN17, PP1, PP8, Psa1, PsP1, PsP2, PsP3, PsP4, PsP5, PS3, PS17, PTB80,PX4, PX7, PYO1, PYO2, PYO5, PYO6, PYO9, PYO1O, PYO13, PYO14, PYO16,PYO18, PYO19, PYO20, PYO29, PYO32, PYO33, PYO35, PYO36, PYO37, PYO38,PYO39, PYO41, PYO42, PYO45, PYO47, PYO48, PYO64, PYO69, PYO103, P1K,SLP1, SL2, S2, UNL-I, wy, Yai, Ya4, Yan, φBE, φCTX, φC17, φKZ,(syn=ΦKZ), φ-LT, Φmu78, φNZ, φPLS-1, φST-1, φW-14, φ-2, 1/72, 2/79, 3,3/DO, 4/237, 5/406, 6C, 6/6660, 7, 7v, 7/184, 8/280, 9/95, 10/502,11/DE, 12/100, 12S, 16, 21, 24, 25F, 27, 31, 44, 68, 71, 95, 109, 188,337, 352, 1214, HN-Pseudomonas (23), A856, B26, CI-I, CI-2, C5, D, gh-1,F1 16, HF, H90, K5, K6, K104, K109, K166, K267, N4, N5, 06N-25P, PE69,Pf, PPN25, PPN35, PPN89, PPN91, PP2, PP3, PP4, PP6, PP7, PP8, PP56,PP87, PP1 14, PP206, PP207, PP306, PP651, Psp231a, Pssy401, Pssy9220,psi, PTB2, PTB20, PTB42, PX1, PX3, PX1O, PX12, PX14, PYO70, PYO71, R,SH6, SH133, tf, Ya5, Ya7, φBS, ΦKf77, φ-MC, ΦmnF82, φPLS27, φPLS743,φS-1, 1, 2, 2, 3, 4, 5, 6, 7, 7, 8, 9, 10, 11, 12, 12B, 13, 14, 15, 14,15, 16, 17, 18, 19, 20, 20, 21, 21, 22, 23, 23, 24, 25, 31, 53, 73,119x, 145, 147, 170, 267, 284, 308, 525, NN-Pseudomonas (5), af, A7, B3,B33, B39, BI-I, C22, D3, D37, D40, D62, D3112, F7, F1O, g, gd, ge, gξHw12, Jb 19, KF1, L^(∘), OXN-32P, O6N-52P, PCH-I, PC13-1, PC35-1, PH2,PH51, PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PM113, PM681, PM682, PO4, PP1, PP4, PP5, PP64, PP65, PP66, PP71, PP86,PP88, PP92, PP401, PP711, PP891, Pssy41, Pssy42, Pssy403, Pssy404,Pssy420, Pssy923, PS4, PS-IO, Pz, SD1, SL1, SL3, SL5, SM, φC5, φC11,φC11-1, φC13, φC15, φMO, φX, φO4, φ1 1, φ240, 2, 2F, 5, 7m, 11, 13,13/441, 14, 20, 24, 40, 45, 49, 61, 73, 148, 160, 198, 218, 222, 236,242, 246, 249, 258, 269, 295, 297, 309, 318, 342, 350, 351, 357-1,400-1, HN-Pseudomonas (6), G1O1, M6, M6a, L1, PB2, Pssy15, Pssy4210,Pssy4220, PYO12, PYO34, PYO49, PYO50, PYO51, PYO52, PYO53, PYO57, PYO59,PYO200, PX2, PX5, SL4, φO3, φO6 and 1214.

Bacteria of the genus Rickettsia are infected by the following phage:NN-Rickettsia.

Bacteria of the genus Salmonella are infected by the following phage: b,Beccles, CT, d, Dundee, f, FeIs 2, GI, GUI, GVI, GVIII, k, K, i, j, L,01, (syn=0-1), (syn=O1), (syn=O-I), (syn=7), 02, 03, P3, P9a, P1O, Sab3,Sab5, San1S, San17, SI, Taunton, ViI, (syn=ViI), 9, imSalmonella (1),N-I, N-5, N-IO, N-17, N-22, 11, 12, 16-19, 20.2, 36, 449C/C178,966A/C259, a, B.A.O.R., e, G4, GUI, L, LP7, M, MG40, N-18, PSA68, P4,P9c, P22, (syn=P22), (syn=PLT22), (syn=PLT22), P22a1, P22-4, P22-7,P22-11, SNT-I, SNT-2, SP6, Vi11i ViIV, ViV, ViVI, ViVII, Worksop, Sj5,ε34, 1, 37, 1(40), (syn=φ1[40]), 1, 422, 2, 2.5, 3b, 4, 5, 6, 14(18), 8,14(6,7), 10, 27, 28B, 30, 31, 32, 33, 34, 36, 37, 39, 1412, SNT-3, 7-11,40.3, c, C236, C557, C625, C966N, g, GV, G5, G1 73, h, IRA, Jersey,MB78, P22-1, P22-3, P22-12, Sab1, Sab2, Sab2, Sab4, San1, San2, San3,San4, San6, San7, San8, San9, San13, San14, San16, San18, San19, San20,San21, San22, San23, San24, San25, San26, SasL1, SasL2, SasL3, SasL4,SasL5, S1BL, SII, ViII, φ1, 1, 2, 3a, 3a1, 1010, Ym-Salmonella (1), N-4,SasL6 and 27.

Bacteria of the genus Serratia are infected by the following phage: A2P,PS20, SMB3, SMP, SMP5, SM2, V40, V56, ic, ΦCP-3, ΦCP-6, 3M, 10/1a, 20A,34CC, 34H, 38T, 345G, 345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4,CW5, Lt232, L2232, L34, L.228, SLP, SMPA, V.43, σ, φCW1, ΦCP6-1, ΦCP6-2,ΦCP6-5, 3T, 5, 8, 9F, 10/1, 2OE, 32/6, 34B, 34CT, 34P, 37, 41, 56, 56D,56P, 6OP, 61/6, 74/6, 76/4, 101/8900, 226, 227, 228, 229F, 286, 289,290F, 512, 764a, 2847/10, 2847/1Oa, L.359 and SMB1.

Bacteria of the genus Shigella are infected by the following phage: Fsa,(syn=a), FSD2d, (syn=D2d), (syn=W2d), FSD2E, (syn=W2e), fv, F6, f7.8,H-Sh, PE5, P90, SfII, Sh, SHm, SHrv, (syn=HIV), SHvi, (syn=HVI), SHVvm,(syn=HVIII), SKγ66, (syn=gamma 66), (syn=γββ), (syn=γ66b), SKm,(syn=SIIIb)5 (syn=UI), SKw, (syn=Siva), (syn=IV), SIC™, (syn=SIVA.),(syn=IVA), SKvi, (syn=KVI), (syn=Svi), (syn=VI), SKvm, (syn=Svm),(syn=VIII), SKVIIIA, (syn=SvmA), (syn=VIIIA), STvi, STK, STx1, STxn,S66, W2, (syn=D2c), (syn=D20), φ1, φIVb 3-SO-R, 8368-SO-R, F7,(syn=FS7), (syn=K29), F1O, (syn=FS1O), (syn=K31), I1, (syn=alfa),(syn=FSa), (syn=K18), (syn=α), I2, (syn=a), (syn=K19), SG33, (syn=G35),(syn=SO-35/G), SG35, (syn=SO-55/G), SG3201, (syn=SO-3201/G), SHn,(syn=HII), SHv, (syn=SHV), SHx, SHX, SKn, (syn=K2), (syn=KII), (syn=Sn),(syn=SsII), (syn=II), SKrv, (syn=Sm), (syn=SsIV), (syn=IV), SK1Va,(syn=Swab), (syn=SsIVa), (syn=IVa), SKV, (syn=K4), (syn=KV), (syn=SV),(syn=SsV), (syn=V), SKx, (syn=K9), (syn=KX), (syn=SX), (syn=SsX),(syn=X), STV, (syn=T35), (syn=35-50-R), STvm, (syn=T8345),(syn=8345-SO-S-R), W1, (syn=D8), (syn=FSD8), W2a, (syn=D2A), (syn=FS2a),DD-2, Sf6, FSi, (syn=F1), SF6, (syn=F6), SG42, (syn=SO-42/G), SG3203,(syn=SO-3203/G), SKF12, (syn=SsF12), (syn=F12), (syn=F12), STn,(syn=1881-SO-R), γ66, (syn=gamma 66a), (syn=Ssγ66), φ2, BI1, DDVII,(syn=DD7), FSD2b, (syn=W2B), FS2, (syn=F2), (syn=F2), FS4, (syn=F4),(syn=F4), FS5, (syn=F5), (syn=F5), FS9, (syn=F9), (syn=F9), FI 1,P2-S0-S, SG36, (syn=SO-36/G), (syn=G36), SG3204, (syn=SO-3204/G),SG3244, (syn=SO-3244/G), SHi, (syn=HI), SHvπ, (syn=HVII), SHK,(syn=HIX), SHx1, SHxπ, (syn=HXn), SKI, KI, (syn=S1), (syn=SsI), SKVII,(syn=KVII), (syn=Svπ), (syn=SsVII), SKIX, (syn=KIX), (syn=S1x),(syn=SsIX), SKXII, (syn=KXII), (syn=Sxn), (syn=SsXII), STi, STff1, STrv,STVi, STvπ, S70, 5206, U2-S0-S, 3210-SO-S, 3859-SO-S, 4020-SO-S, φ3, φ5,φ7, φ8, φ9, φ10, φ11, φ13, φ14, φ18, SHm, (syn=Hπi), SHχi, (syn=HXt) andSKxI, (syn=KXI), (syn=Sχi), (syn=SsXI), (syn=XI).

Bacteria of the genus Staphylococcus are infected by the followingphage: A, EW, K, Ph5, Ph9, PhIO, Ph13, P1, P2, P3, P4, P8, P9, P1O, RG,SB-i, (syn=Sb-I), S3K, Twort, ΦSK311, φ812, 06, 40, 58, 119, 130, 131,200, 1623, STC1, (syn=stc1), STC2, (syn=stc2), 44AHJD, 68, AC1, AC2,A6“C”, A9“C”, b581, CA-I, CA-2, CA-3, CA-4, CA-5, DI 1, L39x35, L54a,M42, N1, N2, N3, N4, N5, N7, N8, N1O, Ni 1, N12, N13, N14, N16, Ph6,Ph12, Ph14, UC-18, U4, U15, S1, S2, S3, S4, S5, X2, Z1, φB5-2, φD, ω,11, (syn=φ11), (syn=P11-M15), 15, 28, 28A, 29, 31, 31B, 37, 42D,(syn=P42D), 44A, 48, 51, 52, 52A, (syn=P52A), 52B, 53, 55, 69, 71,(syn=P71), 71A, 72, 75, 76, 77, 79, 80, 80α, 82, 82A, 83 A, 84, 85, 86,88, 88A, 89, 90, 92, 95, 96, 102, 107, 108, 111, 129-26, 130, 130A, 155,157, 157A, 165, 187, 275, 275A, 275B, 356, 456, 459, 471, 471A, 489,581, 676, 898, 1139, 1154A, 1259, 1314, 1380, 1405, 1563, 2148, 2638A,2638B, 2638C, 2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841, 12100,AC3, A8, A1O, A13, b594n, D, HK2, N9, N15, P52, P87, S1, S6, Z4, φRE,3A, 3B, 3C, 6, 7, 16, 21, 42B, 42C, 42E, 44, 47, 47A5 47C, 51, 54, 54x1,70, 73, 75, 78, 81, 82, 88, 93, 94, 101, 105, 110, 115, 129/16, 174,594n, 1363/14, 2460 and mS-Staphylococcus (1).

Bacteria of the genus Streptococcus are infected by the following phage:EJ-I, NN-Streptococais (1), a, C1, FL0Ths, H39, Cp-I, Cρ-5, Cp-7, Cp-9,Cp-IO, AT298, A5, a1O/J1, a1O/J2, a1O/J5, a1O/J9, A25, BTI 1, b6, CA1,c20-1, c20-2, DP-I, Dp-4, DT1, ET42, e1O, FA101, FEThs, Fκ, FKKIOI,FKLIO, FKP74, FKH, FLOThs, FyIO1, f1, F10, F20140/76, g, GT-234, HB3,(syn=HB-3), HB-623, HB-746, M102, O1205, φO1205, PST, PO, P1, P2, P3,P5, P6, P8, P9, P9, P12, P13, P14, P49, P50, P51, P52, P53, P54, P55,P56, P57, P58, P59, P64, P67, P69, P71, P73, P75, P76, P77, P82, P83,P88, sc, sch, sf, SfI1 1, (syn=SFiI 1), (syn=φSFi11), (syn=ΦSfi1 1),(syn=φSfi1 1), sfi19, (syn=SFi19), (syn=φSFi19), (syn=φSfi19), Sfi21,(syn=SFi21), (syn=φSFi21), (syn=φSfi21), ST0, STX, st2, ST2, ST4, S3,(syn=φS3), s265, Φ17, φ42, Φ57, φ80, φ81, φ82, φ83, φ84, φ85, φ86, φ87,φ88, φ89, φ90, φ91, φ92, φ93, φ94, φ95, φ96, φ97, φ98, φ99, φ100, φ101,φ102, φ227, Φ7201, ω1, ω2, ω3, ω4, ω5, ω6, ω8, ω1O, 1, 6, 9, 1OF, 12/12,14, 17SR, 19S, 24, 50/33, 50/34, 55/14, 55/15, 70/35, 70/36, 71/ST15,71/45, 71/46, 74F, 79/37, 79/38, 80/J4, 80/J9, 80/5T16, 80/15, 80/47,80/48, 101, 103/39, 103/40, 121/41, 121/42, 123/43, 123/44, 124/44,337/ST17 and mStreptococcus (34).

Bacteria of the genus Treponema are infected by the following phage:NN-Treponema (1).

Bacteria of the genus Vibrio are infected by the following phage: CTXΦ,fs, (syn=si), fs2, Ivpf5, Vf12, Vf33, VPIΦ, VSK, v6, 493, CP-T1, ET25,kappa, K139, Labol,)XN-69P, OXN-86, O6N-21P, PB-I, P147, rp-1, SE3,VA-I, (syn=VcA-I), VcA-2, VP1, VP2, VP4, VP7, VP8, VP9, VP1O, VP17,VP18, VP19, X29, (syn=29 d'Herelle), t, ΦHAWI-1, ΦHAWI-2, ΦHAWI-3,ΦHAWI-4, ΦHAWI-5, ΦHAWI-6, ΦHAWI-7, XHAWI-8, ΦHAWI-9, ΦHAWI-10, ΦHCl-1,ΦHC1-2, ΦHC1-3, ΦHC1-4, ΦHC2-1, >HC2-2, ΦHC2-3, ΦHC2-4, ΦHC3-1, ΦHC3-2,ΦHC3-3, ΦHD1S-1, ΦHD1S-2, ΦHD2S-1, ΦHD2S-2, ΦHD2S-3, ΦHD2S-4, ΦHD2S-5,ΦHDO-1, ΦHDO-2, ΦHDO-3, ΦHDO-4, ΦHDO-5, ΦHDO-6, ΦKL-33, ΦKL-34, ΦKL-35,ΦKL-36, ΦKWH-2, ΦKWH-3, ΦKWH-4, ΦMARQ-1, ΦMARQ-2, ΦMARQ-3, ΦMOAT-1,ΦO139, ΦPEL1A-1, ΦPEL1A-2, ΦPEL8A-1, ΦPEL8A-2, ΦPEL8A-3, ΦPEL8C-1,ΦPEL8C-2, ΦPEL13A-1, ΦPEL13B-1, ΦPEL13B-2, ΦPEL13B-3, ΦPEL13B-4,ΦPEL13B-5, ΦPEL13B-6, ΦPEL13B-7, ΦPEL13B-8, ΦPEL13B-9, ΦPEL13B-10,φVP143, φVP253, Φ16, φ138, 1-II, 5, 13, 14, 16, 24, 32, 493, 6214, 7050,7227, II, (syn=group II), (syn==φ2), V, VIII, ˜m-Vibrio (13), KVP20,KVP40, nt-1, O6N-22P, P68, e1, e2, e3, e4, e5, FK, G, I, K, nt-6, N1,N2, N3, N4, N5, O6N-34P, OXN-72P, OXN-85P, OXN-100P, P, Ph-I, PL163/10,Q, S, T, φ92, 1-9, 37, 51, 57, 70A-8, 72A-4, 72A-10, 110A-4, 333, 4996,I (syn=group I), III (syn=group III), VI, (syn=A-Saratov), VII, IX, X,HN-Vibrio (6), pA1, 7, 7-8, 70A-2, 71A-6, 72A-5, 72A-8, 108A-10, 109A-6,109A-8, 1 1OA-1, 110A-5, 110A-7, hv-1, OXN-52P, P13, P38, P53, P65,P108, Pi11, TPI3 VP3, VP6, VP12, VP13, 70A-3, 70A-4, 70A-10, 72A-1,108A-3, 109-B1, 110A-2, 149, (syn=φ149), IV, (syn=group IV), NN-Vibrio(22), VP5, VPI1, VP15, VP16, α1, α2, α3a, α3b, 353B and HN-Vibrio (7).

Bacteria of the genus Yersinia are infected by the following phage: H,H-I, H-2, H-3, H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7,YerA20, YerA41, 3/M64-76, 5/G394-76, 6/C753-76, 8/C239-76, 9/F18167,1701, 1710, PST, 1/F2852-76, D'Herelle, EV, H, Kotljarova, PTB, R, Y,YerA41, φNerO3-12, 3, 4/C1324-76, 7/F783-76, 903, 1/M6176 and Yer2AT.

More preferably, the bacteriophage is selected in the group consistingof Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone,Dickeya virus RC2014, Escherichia virus CBA120, Escherichia virus PhaxI,Salmonella virus 38, Salmonella virus Det7, Salmonella virus GG32,Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus SH19,Salmonella virus SJ3, Escherichia virus ECML4, Salmonella virusMarshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonellavirus STML131, Salmonella virus ViI, Erwinia virus Ea2809, Klebsiellavirus 0507KN21, Serratia virus IME250, Serratia virus MAM1,Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virusCPt10, Campylobacter virus IBB35, Campylobacter virus CP81,Campylobacter virus CP30A, Campylobacter virus CPX, Campylobacter virusNCTC12673, Erwinia virus Ea214, Erwinia virus M7, Escherichia virusAYO145A, Escherichia virus EC6, Escherichia virus HY02, Escherichiavirus JH2, Escherichia virus TP1, Escherichia virus VpaE1, Escherichiavirus wV8, Salmonella virus FelixO1, Salmonella virus HB2014, Salmonellavirus Mushroom, Salmonella virus UAB87, Citrobacter virus Moogle,Citrobacter virus Mordin, Escherichia virus SUSP1, Escherichia virusSUSP2, Aeromonas virus phiO18P, Haemophilus virus HP1, Haemophilus virusHP2, Pasteurella virus F108, Vibrio virus K139, Vibrio virus Kappa,Burkholderia virus phi52237, Burkholderia virus phiE122, Burkholderiavirus phiE202, Escherichia virus 186, Escherichia virus P4, Escherichiavirus P2, Escherichia virus Wphi, Mannheimia virus PHL101, Pseudomonasvirus phiCTX, Ralstonia virus RSA1, Salmonella virus Fe1s2, Salmonellavirus PsP3, Salmonella virus SopEphi, Yersinia virus L413C,Staphylococcus virus G1, Staphylococcus virus G15, Staphylococcus virusJD7, Staphylococcus virus K, Staphylococcus virus MCE2014,Staphylococcus virus P108, Staphylococcus virus Rodi, Staphylococcusvirus 5253, Staphylococcus virus S25-4, Staphylococcus virus SA12,Listeria virus A511, Listeria virus P100, Staphylococcus virus Remus,Staphylococcus virus SA11, Staphylococcus virus Stau2, Bacillus virusCamphawk, Bacillus virus SPO1, Bacillus virus BCP78, Bacillus virusTsarBomba, Staphylococcus virus Twort, Enterococcus virus phiEC24C,Lactobacillus virus Lb338-1, Lactobacillus virus LP65, Enterobactervirus PG7, Escherichia virus CC31, Klebsiella virus JD18, Klebsiellavirus PKO111, Escherichia virus Bp7, Escherichia virus IME08,Escherichia virus JS10, Escherichia virus JS98, Escherichia virus QL01,Escherichia virus VR5, Enterobacter virus Eap3, Klebsiella virus KP15,Klebsiella virus KP27, Klebsiella virus Matisse, Klebsiella virus Miro,Citrobacter virus Merlin, Citrobacter virus Moon, Escherichia virus JSE,Escherichia virus phi1, Escherichia virus RB49, Escherichia virus HX01,Escherichia virus JS09, Escherichia virus RB69, Shigella virus UTAM,Salmonella virus S16, Salmonella virus STML198, Vibrio virus KVP40,Vibrio virus nt1, Vibrio virus Va1KK3, Escherichia virus VR7,Escherichia virus VR20, Escherichia virus VR25, Escherichia virus VR26,Shigella virus SP18, Escherichia virus AR1, Escherichia virus C40,Escherichia virus E112, Escherichia virus ECML134, Escherichia virusHY01, Escherichia virus Ime09, Escherichia virus RB3, Escherichia virusRB14, Escherichia virus T4, Shigella virus Pss1, Shigella virus Shf12,Yersinia virus D1, Yersinia virus PST, Acinetobacter virus 133,Aeromonas virus 65, Aeromonas virus Aeh1, Escherichia virus RB16,Escherichia virus RB32, Escherichia virus RB43, Pseudomonas virus 42,Cronobacter virus CR3, Cronobacter virus CR8, Cronobacter virus CR9,Cronobacter virus PBES02, Pectobacterium virus phiTE, Cronobacter virusGAP31, Escherichia virus 4MG, Salmonella virus SE1, Salmonella virusSSE121, Escherichia virus FFH2, Escherichia virus FV3, Escherichia virusJES2013, Escherichia virus VS, Brevibacillus virus Abouo, Brevibacillusvirus Davies, Bacillus virus Agate, Bacillus virus Bobb, Bacillus virusBp8pC, Erwinia virus Deimos, Erwinia virus Ea35-70, Erwinia virus RAY,Erwinia virus Simmy50, Erwinia virus SpecialG, Acinetobacter virus AB1,Acinetobacter virus AB2, Acinetobacter virus AbC62, Acinetobacter virusAP22, Arthrobacter virus ArV1, Arthrobacter virus Trina, Bacillus virusAvesoBmore, Bacillus virus B4, Bacillus virus Bigbertha, Bacillus virusRiley, Bacillus virus Spock, Bacillus virus Troll, Bacillus virusBastille, Bacillus virus CAM003, Bacillus virus Bc431, Bacillus virusBcp1, Bacillus virus BCP82, Bacillus virus BM15, Bacillus virusDeepblue, Bacillus virus JBP901, Burkholderia virus Bcep1, Burkholderiavirus Bcep43, Burkholderia virus Bcep781, Burkholderia virus BcepNY3,Xanthomonas virus OP2, Burkholderia virus BcepMu, Burkholderia virusphiE255, Aeromonas virus 44RR2, Mycobacterium virus Alice, Mycobacteriumvirus Bxz1, Mycobacterium virus Dandelion, Mycobacterium virus HyRo,Mycobacterium virus 13, Mycobacterium virus Nappy, Mycobacterium virusSebata, Clostridium virus phiC2, Clostridium virus phiCD27, Clostridiumvirus phiCD119, Bacillus virus CP51, Bacillus virus JL, Bacillus virusShanette, Escherichia virus CVM10, Escherichia virus ep3, Erwinia virusAsesino, Erwinia virus EaH2, Pseudomonas virus EL, Halomonas virus HAP1,Vibrio virus VP882, Brevibacillus virus Jimmer, Brevibacillus virusOsiris, Pseudomonas virus Ab03, Pseudomonas virus KPP10, Pseudomonasvirus PAKP3, Sinorhizobium virus M7, Sinorhizobium virus M12,Sinorhizobium virus N3, Erwinia virus Machina, Arthrobacter virus Brent,Arthrobacter virus Jawnski, Arthrobacter virus Martha, Arthrobactervirus Sonny, Edwardsiella virus MSW3, Edwardsiella virus PEi21,Escherichia virus Mu, Shigella virus SfMu, Halobacterium virus phiH,Bacillus virus Grass, Bacillus virus NIT1, Bacillus virus SPG24,Aeromonas virus 43, Escherichia virus P1, Pseudomonas virus CAb1,Pseudomonas virus CAb02, Pseudomonas virus JG004, Pseudomonas virusPAKP1, Pseudomonas virus PAKP4, Pseudomonas virus PaP1, Burkholderiavirus BcepF1, Pseudomonas virus 141, Pseudomonas virus Ab28, Pseudomonasvirus DL60, Pseudomonas virus DL68, Pseudomonas virus F8, Pseudomonasvirus JG024, Pseudomonas virus KPP12, Pseudomonas virus LBL3,Pseudomonas virus LMA2, Pseudomonas virus PB1, Pseudomonas virus SN,Pseudomonas virus PA7, Pseudomonas virus phiKZ, Rhizobium virus RHEph4,Ralstonia virus RSF1, Ralstonia virus RSL2, Ralstonia virus RSL1,Aeromonas virus 25, Aeromonas virus 31, Aeromonas virus Aes12, Aeromonasvirus Aes508, Aeromonas virus AS4, Stenotrophomonas virus IME13,Staphylococcus virus IPLAC1C, Staphylococcus virus SEP1, Salmonellavirus SPN3US, Bacillus virus 1, Geobacillus virus GBSV1, Yersinia virusR1RT, Yersinia virus TG1, Bacillus virus G, Bacillus virus PBS1,Microcystis virus Ma-LMM01, Vibrio virus MAR, Vibrio virus VHML, Vibriovirus VP585, Bacillus virus BPS13, Bacillus virus Hakuna, Bacillus virusMegatron, Bacillus virus WPh, Acinetobacter virus AB3, Acinetobactervirus Abp1, Acinetobacter virus Fri1, Acinetobacter virus IME200,Acinetobacter virus PD6A3, Acinetobacter virus PDAB9, Acinetobactervirus phiAB1, Escherichia virus K30, Klebsiella virus K5, Klebsiellavirus K11, Klebsiella virus Kp1, Klebsiella virus KP32, Klebsiella virusKpV289, Klebsiella virus F19, Klebsiella virus K244, Klebsiella virusKp2, Klebsiella virus KP34, Klebsiella virus KpV41, Klebsiella virusKpV71, Klebsiella virus KpV475, Klebsiella virus SU503, Klebsiella virusSU552A, Pantoea virus Limelight, Pantoea virus Limezero, Pseudomonasvirus LKA1, Pseudomonas virus phiKMV, Xanthomonas virus f20, Xanthomonasvirus f30, Xylella virus Prado, Erwinia virus Era103, Escherichia virusK5, Escherichia virus K1-5, Escherichia virus K1E, Salmonella virus SP6,Escherichia virus T7, Kluyvera virus Kvp1, Pseudomonas virus gh1,Prochlorococcus virus PSSP7, Synechococcus virus P60, Synechococcusvirus Syn5, Streptococcus virus Cp1, Streptococcus virus Cp7,Staphylococcus virus 44AHJD, Streptococcus virus C1, Bacillus virusB103, Bacillus virus GA1, Bacillus virus phi29, Kurthia virus 6,Actinomyces virus Av1, Mycoplasma virus P1, Escherichia virus 24B,Escherichia virus 933W, Escherichia virus Min27, Escherichia virus PA28,Escherichia virus Stx2 II, Shigella virus 7502Stx, Shigella virusPOCJ13, Escherichia virus 191, Escherichia virus PA2, Escherichia virusTL2011, Shigella virus VASD, Burkholderia virus Bcep22, Burkholderiavirus Bcepi102, Burkholderia virus Bcepmig1, Burkholderia virus DC1,Bordetella virus BPP1, Burkholderia virus BcepC6B, Cellulophaga virusCba41, Cellulophaga virus Cba172, Dinoroseobacter virus DFL12, Erwiniavirus Ea9-2, Erwinia virus Frozen, Escherichia virus phiV10, Salmonellavirus Epsilon15, Salmonella virus SPN1S, Pseudomonas virus F116,Pseudomonas virus H66, Escherichia virus APEC5, Escherichia virus APEC7,Escherichia virus Bp4, Escherichia virus EC1UPM, Escherichia virusECBP1, Escherichia virus G7C, Escherichia virus IME11, Shigella virusSb1, Achromobacter virus Axp3, Achromobacter virus JWA1pha, Edwardsiellavirus KF1, Pseudomonas virus KPP25, Pseudomonas virus R18, Pseudomonasvirus Ab09, Pseudomonas virus LIT1, Pseudomonas virus PA26, Pseudomonasvirus Ab22, Pseudomonas virus CHU, Pseudomonas virus LUZ24, Pseudomonasvirus PAA2, Pseudomonas virus PaP3, Pseudomonas virus PaP4, Pseudomonasvirus TL, Pseudomonas virus KPP21, Pseudomonas virus LUZ7, Escherichiavirus N4, Salmonella virus 9NA, Salmonella virus SP069, Salmonella virusBTP1, Salmonella virus HK620, Salmonella virus P22, Salmonella virusST64T, Shigella virus Sf6, Bacillus virus Page, Bacillus virus Palmer,Bacillus virus Pascal, Bacillus virus Pony, Bacillus virus Pookie,Escherichia virus 172-1, Escherichia virus ECB2, Escherichia virus NJ01,Escherichia virus phiEco32, Escherichia virus Septima11, Escherichiavirus SU10, Brucella virus Pr, Brucella virus Tb, Escherichia virusPollock, Salmonella virus FSL SP-058, Salmonella virus FSL SP-076,Helicobacter virus 1961P, Helicobacter virus KHP30, Helicobacter virusKHP40, Hamiltonella virus APSE1, Lactococcus virus KSY1, Phormidiumvirus WMP3, Phormidium virus WMP4, Pseudomonas virus 119X, Roseobactervirus SIO1, Vibrio virus VpV262, Vibrio virus VC8, Vibrio virus VP2,Vibrio virus VP5, Streptomyces virus Amela, Streptomyces virus phiCAM,Streptomyces virus Aaronocolus, Streptomyces virus Caliburn,Streptomyces virus Danzina, Streptomyces virus Hydra, Streptomyces virusIzzy, Streptomyces virus Lannister, Streptomyces virus Lika,Streptomyces virus Sujidade, Streptomyces virus Zemlya, Streptomycesvirus ELB20, Streptomyces virus R4, Streptomyces virus phiHau3,Mycobacterium virus Acadian, Mycobacterium virus Baee, Mycobacteriumvirus Reprobate, Mycobacterium virus Adawi, Mycobacterium virus Bane1,Mycobacterium virus BrownCNA, Mycobacterium virus Chrisnmich,Mycobacterium virus Cooper, Mycobacterium virus JAMaL, Mycobacteriumvirus Nigel, Mycobacterium virus Stinger, Mycobacterium virus Vincenzo,Mycobacterium virus Zemanar, Mycobacterium virus Apizium, Mycobacteriumvirus Manad, Mycobacterium virus Oline, Mycobacterium virus Osmaximus,Mycobacterium virus Pg1, Mycobacterium virus Soto, Mycobacterium virusSuffolk, Mycobacterium virus Athena, Mycobacterium virus Bernardo,Mycobacterium virus Gadjet, Mycobacterium virus Pipefish, Mycobacteriumvirus Godines, Mycobacterium virus Rosebush, Mycobacterium virusBabsiella, Mycobacterium virus Brujita, Mycobacterium virus Che9c,Mycobacterium virus Sbash, Mycobacterium virus Hawkeye, Mycobacteriumvirus Plot, Salmonella virus AG11, Salmonella virus Ent1, Salmonellavirus f18SE, Salmonella virus Jersey, Salmonella virus L13, Salmonellavirus LSPA1, Salmonella virus SE2, Salmonella virus SETP3, Salmonellavirus SETP7, Salmonella virus SETP13, Salmonella virus SP101, Salmonellavirus SS3e, Salmonella virus wks13, Escherichia virus K1G, Escherichiavirus K1H, Escherichia virus K1ind1, Escherichia virus K1ind2,Salmonella virus SP31, Leuconostoc virus Lmd1, Leuconostoc virus LN03,Leuconostoc virus LN04, Leuconostoc virus LN12, Leuconostoc virus LN6B,Leuconostoc virus P793, Leuconostoc virus 1A4, Leuconostoc virus Ln8,Leuconostoc virus Ln9, Leuconostoc virus LN25, Leuconostoc virus LN34,Leuconostoc virus LNTR3, Mycobacterium virus Bongo, Mycobacterium virusRey, Mycobacterium virus Butters, Mycobacterium virus Michelle,Mycobacterium virus Charlie, Mycobacterium virus Pipsqueaks,Mycobacterium virus Xeno, Mycobacterium virus Panchino, Mycobacteriumvirus Phrann, Mycobacterium virus Redi, Mycobacterium virus Skinnyp,Gordonia virus BaxterFox, Gordonia virus Yeezy, Gordonia virus Kita,Gordonia virus Zirinka, Gorrdonia virus Nymphadora, Mycobacterium virusBignuz, Mycobacterium virus Brusacoram, Mycobacterium virus Donovan,Mycobacterium virus Fishburne, Mycobacterium virus Jebeks, Mycobacteriumvirus Malithi, Mycobacterium virus Phayonce, Enterobacter virus F20,Klebsiella virus 1513, Klebsiella virus KLPN1, Klebsiella virus KP36,Klebsiella virus PKP126, Klebsiella virus Sushi, Escherichia virusAHP42, Escherichia virus AHS24, Escherichia virus AKS96, Escherichiavirus C119, Escherichia virus E41c, Escherichia virus Eb49, Escherichiavirus Jk06, Escherichia virus KP26, Escherichia virus Rogue1,Escherichia virus ACGM12, Escherichia virus Rtp, Escherichia virus ADB2,Escherichia virus JMPW1, Escherichia virus JMPW2, Escherichia virus T1,Shigella virus PSf2, Shigella virus Shf11, Citrobacter virus Stevie,Escherichia virus TLS, Salmonella virus SP126, Cronobacter virusEsp2949-1, Pseudomonas virus Ab18, Pseudomonas virus Ab19, Pseudomonasvirus PaMx11, Arthrobacter virus Amigo, Propionibacterium virus Anatole,Propionibacterium virus B3, Bacillus virus Andromeda, Bacillus virusBlastoid, Bacillus virus Curly, Bacillus virus Eoghan, Bacillus virusFinn, Bacillus virus Glittering, Bacillus virus Riggi, Bacillus virusTaylor, Gordonia virus Attis, Mycobacterium virus Barnyard,Mycobacterium virus Konstantine, Mycobacterium virus Predator,Mycobacterium virus Bernal13, Staphylococcus virus 13, Staphylococcusvirus 77, Staphylococcus virus 108PVL, Mycobacterium virus Bron,Mycobacterium virus Faith1, Mycobacterium virus Joedirt, Mycobacteriumvirus Rumpelstiltskin, Lactococcus virus bIL67, Lactococcus virus c2,Lactobacillus virus c5, Lactobacillus virus Ld3, Lactobacillus virusLd17, Lactobacillus virus Ld25A, Lactobacillus virus LLKu, Lactobacillusvirus phiLdb, Cellulophaga virus Cba121, Cellulophaga virus Cba171,Cellulophaga virus Cba181, Cellulophaga virus ST, Bacillus virus 250,Bacillus virus IEBH, Mycobacterium virus Ardmore, Mycobacterium virusAvani, Mycobacterium virus Boomer, Mycobacterium virus Che8,Mycobacterium virus Che9d, Mycobacterium virus Deadp, Mycobacteriumvirus Dlane, Mycobacterium virus Dorothy, Mycobacterium virusDotproduct, Mycobacterium virus Drago, Mycobacterium virus Fruitloop,Mycobacterium virus Gumbie, Mycobacterium virus Ibhubesi, Mycobacteriumvirus Llij, Mycobacterium virus Mozy, Mycobacterium virus Mutaforma13,Mycobacterium virus Pacc40, Mycobacterium virus PMC, Mycobacterium virusRamsey, Mycobacterium virus Rockyhorror, Mycobacterium virus SG4,Mycobacterium virus Shaunal, Mycobacterium virus Shilan, Mycobacteriumvirus Spartacus, Mycobacterium virus Taj, Mycobacterium virus Tweety,Mycobacterium virus Wee, Mycobacterium virus Yoshi, Salmonella virusChi, Salmonella virus FSLSP030, Salmonella virus FSLSP088, Salmonellavirus iEPS5, Salmonella virus SPN19, Mycobacterium virus 244,Mycobacterium virus Bask21, Mycobacterium virus CJW1, Mycobacteriumvirus Eureka, Mycobacterium virus Kostya, Mycobacterium virus Porky,Mycobacterium virus Pumpkin, Mycobacterium virus Sirduracell,Mycobacterium virus Toto, Mycobacterium virus Corndog, Mycobacteriumvirus Firecracker, Rhodobacter virus RcCronus, Pseudomonas virus D3112,Pseudomonas virus DMS3, Pseudomonas virus FHA0480, Pseudomonas virusLPB1, Pseudomonas virus MP22, Pseudomonas virus MP29, Pseudomonas virusMP38, Pseudomonas virus PA1KOR, Pseudomonas virus D3, Pseudomonas virusPMG1, Arthrobacter virus Decurro, Gordonia virus Demosthenes, Gordoniavirus Katyusha, Gordonia virus Kvothe, Propionibacterium virus B22,Propionibacterium virus Doucette, Propionibacterium virus E6,Propionibacterium virus G4, Burkholderia virus phi6442, Burkholderiavirus phi1026b, Burkholderia virus phiE125, Edwardsiella virus eiAU,Mycobacterium virus Ff47, Mycobacterium virus Muddy, Mycobacterium virusGaia, Mycobacterium virus Giles, Arthrobacter virus Captnmurica,Arthrobacter virus Gordon, Gordonia virus GordTnk2, Paenibacillus virusHarrison, Escherichia virus EK99P1, Escherichia virus HK578, Escherichiavirus JL1, Escherichia virus SSL2009a, Escherichia virus YD2008s,Shigella virus EP23, Sodalis virus SO1, Escherichia virus HK022,Escherichia virus HK75, Escherichia virus HK97, Escherichia virus HK106,Escherichia virus HK446, Escherichia virus HK542, Escherichia virusHK544, Escherichia virus HK633, Escherichia virus mEp234, Escherichiavirus mEp235, Escherichia virus mEpX1, Escherichia virus mEpX2,Escherichia virus mEp043, Escherichia virus mEp213, Escherichia virusmEp237, Escherichia virus mEp390, Escherichia virus mEp460, Escherichiavirus mEp505, Escherichia virus mEp506, Brevibacillus virus Jenst,Achromobacter virus 83-24, Achromobacter virus JWX, Arthrobacter virusKellezzio, Arthrobacter virus Kitkat, Arthrobacter virus Bennie,Arthrobacter virus DrRobert, Arthrobacter virus Glenn, Arthrobactervirus HunterDalle, Arthrobacter virus Joann, Arthrobacter virus Korra,Arthrobacter virus Preamble, Arthrobacter virus Pumancara, Arthrobactervirus Wayne, Mycobacterium virus Alma, Mycobacterium virus Arturo,Mycobacterium virus Astro, Mycobacterium virus Backyardigan,Mycobacterium virus BBPiebs31, Mycobacterium virus Benedict,Mycobacterium virus Bethlehem, Mycobacterium virus Billknuckles,Mycobacterium virus Bruns, Mycobacterium virus Bxb1, Mycobacterium virusBxz2, Mycobacterium virus Che12, Mycobacterium virus Cuco, Mycobacteriumvirus D29, Mycobacterium virus Doom, Mycobacterium virus Ericb,Mycobacterium virus Euphoria, Mycobacterium virus George, Mycobacteriumvirus Gladiator, Mycobacterium virus Goose, Mycobacterium virus Hammer,Mycobacterium virus Heldan, Mycobacterium virus Jasper, Mycobacteriumvirus JC27, Mycobacterium virus Jeffabunny, Mycobacterium virus JHC117,Mycobacterium virus KBG, Mycobacterium virus Kssjeb, Mycobacterium virusKuge1, Mycobacterium virus L5, Mycobacterium virus Lesedi, Mycobacteriumvirus LHTSCC, Mycobacterium virus lockley, Mycobacterium virus Marcell,Mycobacterium virus Microwolf, Mycobacterium virus Mrgordo,Mycobacterium virus Museum, Mycobacterium virus Nepal, Mycobacteriumvirus Packman, Mycobacterium virus Peaches, Mycobacterium virus Perseus,Mycobacterium virus Pukovnik, Mycobacterium virus Rebeuca, Mycobacteriumvirus Redrock, Mycobacterium virus Ridgecb, Mycobacterium virusRockstar, Mycobacterium virus Saintus, Mycobacterium virus Skipole,Mycobacterium virus Solon, Mycobacterium virus Switzer, Mycobacteriumvirus SWU1, Mycobacterium virus Ta17a, Mycobacterium virus Tiger,Mycobacterium virus Timshe1, Mycobacterium virus Trixie, Mycobacteriumvirus Turbido, Mycobacterium virus Twister, Mycobacterium virus U2,Mycobacterium virus Violet, Mycobacterium virus Wonder, Escherichiavirus DE3, Escherichia virus HK629, Escherichia virus HK630, Escherichiavirus Lambda, Arthrobacter virus Laroye, Mycobacterium virus Halo,Mycobacterium virus Liefie, Mycobacterium virus Marvin, Mycobacteriumvirus Mosmoris, Arthrobacter virus Circum, Arthrobacter virus Mudcat,Escherichia virus N15, Escherichia virus 9g, Escherichia virus JenK1,Escherichia virus JenP1, Escherichia virus JenP2, Pseudomonas virus NP1,Pseudomonas virus PaMx25, Mycobacterium virus Baka, Mycobacterium virusCourthouse, Mycobacterium virus Littlee, Mycobacterium virus Omega,Mycobacterium virus Optimus, Mycobacterium virus Thibault, Polaribactervirus P12002L, Polaribacter virus P12002S, Nonlabens virus P12024L,Nonlabens virus P12024S, Thermus virus P23-45, Thermus virus P74-26,Listeria virus LP26, Listeria virus LP37, Listeria virus LP110, Listeriavirus LP114, Listeria virus P70, Propionibacterium virus ATCC29399BC,Propionibacterium virus ATCC29399BT, Propionibacterium virus Attacne,Propionibacterium virus Keiki, Propionibacterium virus Kubed,Propionibacterium virus Lauchelly, Propionibacterium virus MrAK,Propionibacterium virus Ouroboros, Propionibacterium virus P91,Propionibacterium virus P105, Propionibacterium virus P144,Propionibacterium virus P1001, Propionibacterium virus P1.1,Propionibacterium virus P100A, Propionibacterium virus P100D,Propionibacterium virus P101A, Propionibacterium virus P104A,Propionibacterium virus PA6, Propionibacterium virus Pacnes201215,Propionibacterium virus PAD20, Propionibacterium virus PAS50,Propionibacterium virus PHL009M11, Propionibacterium virus PHL025M00,Propionibacterium virus PHL037M02, Propionibacterium virus PHL041M10,Propionibacterium virus PHL060L00, Propionibacterium virus PHL067M01,Propionibacterium virus PHL070N00, Propionibacterium virus PHL071N05,Propionibacterium virus PHL082M03, Propionibacterium virus PHL092M00,Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01,Propionibacterium virus PHL112N00, Propionibacterium virus PHL113M01,Propionibacterium virus PHL114L00, Propionibacterium virus PHL116M00,Propionibacterium virus PHL117M00, Propionibacterium virus PHL117M01,Propionibacterium virus PHL132N00, Propionibacterium virus PHL141N00,Propionibacterium virus PHL151M00, Propionibacterium virus PHL151N00,Propionibacterium virus PHL152M00, Propionibacterium virus PHL163M00,Propionibacterium virus PHL171M01, Propionibacterium virus PHL179M00,Propionibacterium virus PHL194M00, Propionibacterium virus PHL199M00,Propionibacterium virus PHL301M00, Propionibacterium virus PHL308M00,Propionibacterium virus Pirate, Propionibacterium virus Procrass1,Propionibacterium virus SKKY, Propionibacterium virus Solid,Propionibacterium virus Stormborn, Propionibacterium virus Wizzo,Pseudomonas virus PaMx28, Pseudomonas virus PaMx74, Mycobacterium virusPatience, Mycobacterium virus PBI1, Rhodococcus virus Pepy6, Rhodococcusvirus Poco6, Propionibacterium virus PFR1, Streptomyces virus phiBT1,Streptomyces virus phiC31, Streptomyces virus TG1, Caulobacter virusKarma, Caulobacter virus Magneto, Caulobacter virus phiCbK, Caulobactervirus Rogue, Caulobacter virus Swift, Staphylococcus virus 11,Staphylococcus virus 29, Staphylococcus virus 37, Staphylococcus virus53, Staphylococcus virus 55, Staphylococcus virus 69, Staphylococcusvirus 71, Staphylococcus virus 80, Staphylococcus virus 85,Staphylococcus virus 88, Staphylococcus virus 92, Staphylococcus virus96, Staphylococcus virus 187, Staphylococcus virus 52a, Staphylococcusvirus 80alpha, Staphylococcus virus CNPH82, Staphylococcus virus EW,Staphylococcus virus IPLA5, Staphylococcus virus IPLA7, Staphylococcusvirus IPLA88, Staphylococcus virus PH15, Staphylococcus virus phiETA,Staphylococcus virus phiETA2, Staphylococcus virus phiETA3,Staphylococcus virus phiMR11, Staphylococcus virus phiMR25,Staphylococcus virus phiNM1, Staphylococcus virus phiNM2, Staphylococcusvirus phiNM4, Staphylococcus virus SAP26, Staphylococcus virus X2,Enterococcus virus FL1, Enterococcus virus FL2, Enterococcus virus FL3,Lactobacillus virus ATCC8014, Lactobacillus virus phiJL1, Pediococcusvirus cIP1, Aeromonas virus pIS4A, Listeria virus LP302, Listeria virusPSA, Methanobacterium virus psiM1, Roseobacter virus RDJL1, Roseobactervirus RDJL2, Rhodococcus virus RER2, Enterococcus virus BC611,Enterococcus virus IMEEF1, Enterococcus virus SAP6, Enterococcus virusVD13, Streptococcus virus SPQS1, Mycobacterium virus Papyrus,Mycobacterium virus Send513, Burkholderia virus KL1, Pseudomonas virus73, Pseudomonas virus Ab26, Pseudomonas virus Kakheti25, Escherichiavirus Cajan, Escherichia virus Seurat, Staphylococcus virus SEP9,Staphylococcus virus Sextaec, Streptococcus virus 858, Streptococcusvirus 2972, Streptococcus virus ALQ132, Streptococcus virus 01205,Streptococcus virus Sfi11, Streptococcus virus 7201, Streptococcus virusDT1, Streptococcus virus phiAbc2, Streptococcus virus Sfi19,Streptococcus virus Sfi21, Paenibacillus virus Diva, Paenibacillus virusHb10c2, Paenibacillus virus Rani, Paenibacillus virus Shelly,Paenibacillus virus Sitara, Paenibacillus virus Willow, Lactococcusvirus 712, Lactococcus virus ASCC191, Lactococcus virus ASCC273,Lactococcus virus ASCC281, Lactococcus virus ASCC465, Lactococcus virusASCC532, Lactococcus virus Bibb29, Lactococcus virus bIL170, Lactococcusvirus CB13, Lactococcus virus CB14, Lactococcus virus CB19, Lactococcusvirus CB20, Lactococcus virus jj50, Lactococcus virus P2, Lactococcusvirus P008, Lactococcus virus sk1, Lactococcus virus S14, Bacillus virusSlash, Bacillus virus Stahl, Bacillus virus Staley, Bacillus virusStills, Gordonia virus Bachita, Gordonia virus ClubL, Gordonia virusOneUp, Gordonia virus Smoothie, Gordonia virus Soups, Bacillus virusSPbeta, Vibrio virus MAR10, Vibrio virus SSP002, Escherichia virusAKFV33, Escherichia virus BF23, Escherichia virus DT57C, Escherichiavirus EPS7, Escherichia virus FFH1, Escherichia virus H8, Escherichiavirus slur09, Escherichia virus T5, Salmonella virus 118970sa12,Salmonella virus Shivani, Salmonella virus SPC35, Salmonella virusStitch, Arthrobacter virus Tank, Tsukamurella virus TIN2, Tsukamurellavirus TIN3, Tsukamurella virus TIN4, Rhodobacter virus RcSpartan,Rhodobacter virus RcTitan, Mycobacterium virus Anaya, Mycobacteriumvirus Angelica, Mycobacterium virus Crimd, Mycobacterium virusFionnbarth, Mycobacterium virus Jaws, Mycobacterium virus Larva,Mycobacterium virus Macncheese, Mycobacterium virus Pixie, Mycobacteriumvirus TM4, Bacillus virus BMBtp2, Bacillus virus TP21, Geobacillus virusTp84, Staphylococcus virus 47, Staphylococcus virus 3a, Staphylococcusvirus 42e, Staphylococcus virus IPLA35, Staphylococcus virus phi12,Staphylococcus virus phiSLT, Mycobacterium virus 32HC, Rhodococcus virusRGL3, Paenibacillus virus Vegas, Gordonia virus Vendetta, Bacillus virusWbeta, Mycobacterium virus Wildcat, Gordonia virus Twister6, Gordoniavirus Wizard, Gordonia virus Hotorobo, Gordonia virus Monty, Gordoniavirus Woes, Xanthomonas virus CP1, Xanthomonas virus OP1, Xanthomonasvirus phi17, Xanthomonas virus Xop411, Xanthomonas virus Xp10,Streptomyces virus TP1604, Streptomyces virus YDN12, Alphaproteobacteriavirus phiJ1001, Pseudomonas virus LKO4, Pseudomonas virus M6,Pseudomonas virus MP1412, Pseudomonas virus PAE1, Pseudomonas virus Yua,Pseudoalteromonas virus PM2, Pseudomonas virus phi6, Pseudomonas virusphi8, Pseudomonas virus phi12, Pseudomonas virus phi13, Pseudomonasvirus phi2954, Pseudomonas virus phiNN, Pseudomonas virus phiYY, Vibriovirus fs1, Vibrio virus VGJ, Ralstonia virus RS603, Ralstonia virusRSM1, Ralstonia virus RSM3, Escherichia virus M13, Escherichia virus122, Salmonella virus IKe, Acholeplasma virus L51, Vibrio virus fs2,Vibrio virus VFJ, Escherichia virus If1, Propionibacterium virus B5,Pseudomonas virus Pf1, Pseudomonas virus Pf3, Ralstonia virus PE226,Ralstonia virus RSS1, Spiroplasma virus SVTS2, Stenotrophomonas virusPSH1, Stenotrophomonas virus SMA6, Stenotrophomonas virus SMA7,Stenotrophomonas virus SMA9, Vibrio virus CTXphi, Vibrio virus KSF1,Vibrio virus VCY, Vibrio virus Vf33, Vibrio virus VfO3K6, Xanthomonasvirus Cf1c, Spiroplasma virus C74, Spiroplasma virus R8A2B, Spiroplasmavirus SkV1CR23x, Escherichia virus FI, Escherichia virus Qbeta,Escherichia virus BZ13, Escherichia virus MS2, Escherichia virus alpha3,Escherichia virus ID21, Escherichia virus ID32, Escherichia virus ID62,Escherichia virus NC28, Escherichia virus NC29, Escherichia virus NC35,Escherichia virus phiK, Escherichia virus St1, Escherichia virus WA45,Escherichia virus G4, Escherichia virus ID52, Escherichia virus Talmos,Escherichia virus phiX174, Bdellovibrio virus MAC1, Bdellovibrio virusMH2K, Chlamydia virus Chp1, Chlamydia virus Chp2, Chlamydia virusCPAR39, Chlamydia virus CPG1, Spiroplasma virus SpV4, Acholeplasma virusL2, Pseudomonas virus PR4, Pseudomonas virus PRD1, Bacillus virus AP50,Bacillus virus Bam35, Bacillus virus GIL16, Bacillus virus Wip1,Escherichia virus phi80, Escherichia virus RB42, Escherichia virus T2,Escherichia virus T3, Escherichia virus T6, Escherichia virus VT2-Sa,Escherichia virus VT1-Sakai, Escherichia virus VT2-Sakai, Escherichiavirus CP-933V, Escherichia virus P27, Escherichia virus Stx2phi-I,Escherichia virus Stx1phi, Escherichia virus Stx2phi-II, Escherichiavirus CP-1639, based on the Escherichia virus BP-4795, Escherichia virus86, Escherichia virus Min27, Escherichia virus 2851, Escherichia virus1717, Escherichia virus YYZ-2008, Escherichia virus EC026_P06,Escherichia virus ECO103_P15, Escherichia virus ECO103_P12, Escherichiavirus ECO111_P16, Escherichia virus ECO111_P11, Escherichia virusVT2phi_272, Escherichia virus TL-2011c, Escherichia virus P13374,Escherichia virus Spy.

In one embodiment, the bacterial virus particles target E. coli andincludes the capsid of a bacteriophage selected in the group consistingof BW73, B278, D6, D108, E, E1, E24, E41, FI-2, FI-4, FI-5, HI8A, Ff18B,i, MM, Mu, 025, PhI-5, Pk, PSP3, P1, P1D, P2, P4, S1, Wφ, φK13, φ1, φ2,φ7, φ92, 7 A, 8φ, 9φ, 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM,C4, C16, DD-VI, E4, E7, E28, FI1, FI3, H, H1, H3, H8, K3, M, N, ND-2,ND-3, ND4, ND-5, ND6, ND-7, Ox-I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, PhI-I,RB42, RB43, RB49, RB69, S, SaI-I, Sa1-2, Sa1-3, Sa1-4, Sa1-5, Sa1-6,TC23, TC45, TuII*-6, TuIP-24, TuII*46, TuIP-60, T2, T4, T6, T35, α1, 1,IA, 3, 3A, 3T+, 5φ, 9266Q, CFO103, HK620, J, K, K1F, m59, no. A, no. E,no. 3, no. 9, N4, sd, T3, T7, WPK, W31, ΔH, φC3888, φK3, φK7, φK12,φV-1, Φ04-CF, Φ05, Φ06, Φ07, φ1, φ1.2, φ20, φ95, φ263, φ1O92, φ1, φ11,Ω8, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W,NN-Escherichia (1), Esc-7-11, AC30, CVX-5, C1, DDUP, EC1, EC2, E21, E29,F1, F26S, F27S, Hi, HK022, HK97, HK139, HK253, HK256, K7, ND-I, PA-2, q,S2, T1,), T3C, T5, UC-I, w, β4, γ2, λ, ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ,2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50,AC57, AC81, AC95, HK243, K1O, ZG/3A, 5, 5A, 21EL, H19-J and 933H.

Bacterial Delivery Vehicle

In one aspect, the bacterial delivery vehicle is a bacterial virusparticle. In such embodiment, the payload may be a plasmid.Particularly, the invention may concern a bacterial virus particle withthe plasmid according to the invention as disclosed hereabove (inparticular it comprises at least two packaging sites) encapsidated intothe particle. It also relates to a combination of at least two differentbacterial virus particles, said different bacterial virus particleshaving the same plasmid encapsidated into the particles.

The different bacterial delivery vehicles are capable of targeting atleast two different bacteria and of introducing the plasmid into saidbacteria. Alternatively, the different bacterial virus particles arecapable of targeting the same bacteria and of introducing the payloadinto said bacteria. The spectra of the population of bacterial virusparticles is defined according to the bacteriophages infection spectra,as describe hereabove.

The particles may comprise the capsid of bacteriophages selected of inthe group consisting of lambda derived capsids, P4 derived capsids,M13-derived capsids, P1 derived capsids (see, e.g., Westwater C A etal., Microbiology 148, 943-50 (2002); Kittleson J T et al., ACSSynthetoc Biology 1, 583-89 (2012); Mead D A et al, Biotechnology 10,85-102 (1988)).

Optionally, the phagemid is selected from the group consisting of lambdaderived phagemids, P4 derived phagemids, M13-derived phagemids, such asthe ones containing the fl origin for filamentous phage packaging suchas, for example, pBluescript II SK (+/−) and KS (+/−) phagemids, pBC SKand KS phagemids, pADL and P1 derived phagemids, preferably phagemidsaccording to the invention are selected from lambda derived phagemidsand P4 derived phagemids, more preferably, phagemids according to theinvention are selected from lambda derived phagemids, preferablyselected from the group consisting of HK022 derived phagemids, mEP237derived phagemids, HK97 derived phagemids, HK629 derived phagemids,HK630 derived phagemids, mEP043 derived phagemids, mEP213 derivedphagemids, mEP234 derived phagemids, mEP390 derived phagemids, mEP460derived phagemids, mEPx1 derived phagemids, mEPx2 derived phagemids,phi80 derived phagemids and mEP234 derived phagemids.

In a preferred embodiment, the bacterial delivery vehicles comprise thecapsid of bacteriophages selected in the group consisting of P2, P4, λ,and 186.

In an even more preferred embodiment, the bacterial delivery vehiclescomprise the capsid of bacteriophages P2 and λ.

More preferably, the bacterial delivery vehicles may comprise the capsidof bacteriophages selected from lambda derived capsids, preferablyselected from the group consisting of HK022 derived capsids, mEP237derived capsids, HK97 derived capsids, HK629 derived capsids, HK630derived capsids, mEP043 derived capsids, mEP213 derived capsids, mEP234derived capsids, mEP390 derived capsids, mEP460 derived capsids, mEPx1derived capsids, mEPx2 derived capsids, phi80 derived capsids, mEP234derived capsids.

Method for Preparing the Bacterial Virus Particles—Producing Bacteria

In another aspect, a method is provided for preparing a population of atleast two different bacterial virus particles containing the samepayload.

In this particular aspect, the method comprises the introduction of thepayload according to the invention into bacteria, the productionbacteria. For instance, in the embodiment where the bacterial deliveryvehicle are bacterial virus particles, the bacteria can be infected(i.e. according to the bacteriophage spectra known by the person skilledin the art). Alternatively, the bacteria can be transfected by thepayload according to the invention.

The bacterium is suitable for the replication of the payload accordingto the invention and its packaging into at least one of the differentbacterial delivery vehicles. For this purpose, the bacteria may furthercomprise satellite or helper phages genes to promote the packaging ofthe payload. The bacteria express the structural and functional proteinsnecessary to promote an in vitro packaging of the payload in a bacterialdelivery vehicle, particularly in a bacterial virus particle. Inparticular, the bacteria express the protein of the particle, namely thecapsid or coat proteins.

In one aspect, a first bacterium is used for producing the payloadpackaged into a first bacterial delivery vehicle and a second bacteriumis used for producing the same payload packaged into a second bacterialvirus particle. Then, for each bacterial virus particle, a specificbacterium is used for the production of the payload packaged into aparticular bacterial virus particle. Accordingly, each specificbacterium comprises satellite or helper phage genes to promote thepackaging of the payload into the particular bacterial virus particle.In particular, the first bacterium expresses the structural andfunctional proteins necessary to promote an intracellular (i.e.intrabacterial) packaging of the payload into a first bacterial virusparticle; and the second bacterium expresses the structural andfunctional proteins necessary to promote an intracellular packaging ofthe payload into a second bacterial virus particle. In one embodiment,the first bacterium expresses the capsid or coat proteins of a firstbacterial virus particle; and the second bacterium expresses the capsidor coat proteins of a second bacterial virus particle. Of course, ifmore than two bacterial virus particles are contemplated, the sameapplies to the additional bacterial virus particles. The presentinvention also relates to a kit comprising such a first and secondbacteria and so on.

In another aspect, one bacterium is used for producing the payloadpackaged into a first bacterial delivery vehicle and a second bacterialdelivery vehicle. Then, for a combination of at least two bacterialdelivery vehicles, a bacterium is used for the production of the payloadpackaged into at least two different bacterial delivery vehicles.Accordingly, a bacterium comprises satellite or helper phages genes topromote the packaging of the payload into at least two differentbacterial delivery vehicles, preferably into at least two differentbacterial virus particles. In particular, the bacterium expresses thestructural and functional proteins necessary to promote an intracellularpackaging of the payload into a first bacterial delivery vehicle and asecond bacterial virus particle. In one embodiment, the bacteriumexpresses the capsid or coat proteins of a first bacterial virusparticle and of a second bacterial virus particle. Of course, if morethan two bacterial virus particles are contemplated, the same applies tothe additional bacterial virus particles.

Then, the method comprises:

-   -   providing the payload as defined above;    -   introducing said payload into bacteria, the bacteria being        suitable for packaging the payload into a bacterial delivery        vehicle; and    -   packaging the payload into the bacterial delivery vehicles.

The method may further comprise a step of recovering the bacterialdelivery vehicles having the payload packaged into them.

The introduction of the payload into bacteria can be carried out bytransfection or by injection.

Such methods may further comprise the use of a helper phage to promotethe packaging in at least two different delivery vehicles. It is knownby the person skilled in the art that some bacteriophages are defectiveand need a helper phage for replication and/or packaging. Thus,compatible pairs of principal phage and helper phage are easily made bythe person skilled in the art to promote an efficient payload packaging.Such Helper phage can be but are not limited to M13KO7, R408, VCSM13,KM13 (Res Microbiol (2001) 152, 187-191), M13MDD3.2 (FEMS Microbiol Lett(1995) 125, 317-321), R408d3 (Gene (1997) 198, 99-103), VCSM13d3 (Gene(1997) 198, 99-103), Hyperphage (Nat Biotechnol (2001) 19, 75-78), CThelper phage (Nucleic Acids Res (2003) 31, e59), Ex-phage (Nucleic AcidsRes (2002) 30, e18), Phaberge (J Immunol Methods (2003) 274, 233-244),XP5 (J Immunol Methods (2012) 376, 46-54), DeltaPhage (Nucleic Acids Res(2012) 40, e120). A bacterial strain carrying a helper phage derivativethat expresses all the components required for encapsidation may also beused.

In another particular embodiment, such methods further comprise the useof a satellite phage. Preferably, the satellite phages can encodeproteins that promote capsid size reduction of the principal phage, asdescribed for the P4 Sid protein that controls the P2 capsid size to fitits smaller genome. Such satellite phage can for example be phages P4.

In another aspect, such methods further comprise the use of P4 satellitephage proteins, preferably the Sid protein, to promote the encapsidationof the payload in P2 bacterial virus capsid.

Alternatively, the bacterial delivery vehicle (i.e. bacterial virusparticle) can be produced in vitro by contacting the payload with thestructural and functional proteins necessary to promote an in vitropackaging of the payload into a particular bacterial virus particle(Hohn et al, PNAS, 1977)(Collins et al, PNAS, 1978)(Gunther et al, NAR,1993)

Kit and Uses Thereof

In some embodiments, the present invention also relates to a kitcomprising the payload as disclosed herein, optionally a satellite phageand/or a helper phage to promote the packaging of the payload in the atleast two bacteria delivery vehicles and optionally bacterial cellssuitable for packaged payload production.

In one embodiment, the kit further comprises a satellite phage and/or ahelper phage to promote the packaging of the payload in the at least twobacteria delivery vehicles.

In a particular embodiment, the kit further comprises a Helper phageselected in the group consisting of M13KO7, R408, VCSM13, KM13 (ResMicrobiol (2001) 152, 187-191), M13MDD3.2 (FEMS Microbiol Lett (1995)125, 317-321), R408d3 (Gene (1997) 198, 99-103), VCSM13d3 (Gene (1997)198, 99-103), Hyperphage (Nat Biotechnol (2001) 19, 75-78), CT helperphage (Nucleic Acids Res (2003) 31, e59), Ex-phage (Nucleic Acids Res(2002) 30, e18), Phaberge (J Immunol Methods (2003) 274, 233-244), XP5(J Immunol Methods (2012) 376, 46-54), DeltaPhage (Nucleic Acids Res(2012) 40, e120).

In another embodiment, the kit further comprises a satellite phage orsatellite phage genes. Preferably, the satellite phages can encodeproteins that promote capsid size reduction of the principal phage, asdescribed for the P4 Sid protein that controls the P2 capsid size to fitits smaller genome.

In some embodiments, the kit further comprises vials containing naturalor non-natural bacterial host cells suitable for packaged payloadproduction.

In one embodiment, the kit further comprises bacterial cells suitablefor packaged payload production selected in the group consisting ofActinomyces, Achromobacter, Acidaminococcus, Acinetobacter, Aeromonas,Alcaligenes, Bacillus, Bacteroides, Bifidobacterium, Bordetella,Borrelia, Brucella, Burkholderia, Butyriviberio, Campylobacter,Chlamydia, Citrobacter, Clostridium, Corynebacterium, Eikenella,Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Eubacterium,Flavobacterium, Francisella, Fusobacterium, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria,Methanobrevibacter, Morganella, Mycobacterium, Mycoplamsa, Neisseria,Nocardia, Peptococcus, Prevotella, Providencia, Propionibacterium,Plesiomonas, Pseudomonas, Rickettsia, Ruminococcus, Rhodococcus,Salmonella, Sarcina, Serratia, Shigella, Spirillum, StaphylococcusStreptobacillus, Streptococcus, Treponema, Vibrio and Yersinia.

In one embodiment the bacteria carry a helper phage or a satellite phagederivative that expresses all the components required for the payloadpackaging.

In certain embodiments, the bacterial host cells are E. coli.

The kit may further comprise one or more of wash buffers and/orreagents, hybridization buffers and/or reagents, labelling buffersand/or reagents, and detection means. The buffers and/or reagents areusually optimized for the particular utilization for which the kit isintended. Protocols for using these buffers and reagents for performingdifferent steps of the procedure may also be included in the kit.Further optional components of the kits may include expression mediawith different supply (antibiotics or nutriment) for bacteria growthand/or selection of bacteria containing the payload of the invention.

Pharmaceutical or Veterinary Composition

The present invention relates to a pharmaceutical or veterinarycomposition comprising the payload as described hereabove packaged intoa bacterial delivery vehicle. More particularly, the present inventionrelates to a pharmaceutical or veterinary composition comprising thepayload described hereabove packaged into at least two differentbacterial delivery vehicles.

Accordingly, the present invention relates to a pharmaceutical orveterinary composition comprising at least two different bacterial virusparticles with the same payload as described hereabove packaged intothem. The at least two different bacterial virus particles can beprepared from any of the above described bacteriophages.

In one embodiment, the pharmaceutical composition comprising at leasttwo bacterial virus particles capable of targeting at least twodifferent bacteria and of introducing the payload into the bacteria.Alternatively, the pharmaceutical composition comprising at least twobacterial virus particles capable of targeting the same bacterium and ofintroducing the payload into this bacterium.

In some embodiment, the pharmaceutical composition comprises severalbacterial delivery vehicles with the same payload according to thepresent invention and/or bacterial delivery vehicles with differentpayload inside.

For pharmaceutical composition comprising different bacterial deliveryvehicles with the same payload inside, it can be also reallyadvantageous to be able to identify and quantify in the composition,each of the bacterial delivery vehicles individually. Even if thecomposition comprises bacterial delivery vehicles or bacterial virusparticles which are different, they may share common conserved regions,which makes it difficult to develop protein detection and quantificationassays (ELISA, Western Blot) specific enough to reliably detect andquantify each delivery vehicle or virus particle individually from thecomposition. To overcome this hurdle which can be of importance from theregulatory standpoint, a cocktail or mixture of bacterial deliveryvehicles can be developed that contain identical DNA payloads with theexception of a unique “tracer” sequence designed in such a way that allthe payloads packaged in a same delivery vehicles have the same “tracer”sequence and payloads packaged in different delivery vehicles havedifferent “tracer” sequences. In this way, both the presence and therelative abundance of each delivery vehicle in a mixture can beconfirmed by PCR, qPCR, ddPCR, or NGS using primers specific for the“tracer” sequences. In a preferred aspect, the tracer is composed of twonucleic acid constant regions flanking one nucleic acid variable region.Each region of the tracer can be of any length, and more preferablybetween 25 and 50 nucleotides each.

Alternatively, the mixture of delivery vehicles contains deliveryvehicles comprising different DNA payloads, each DNA payload comprisingdifferent tracer or the same tracer, in order to allow detection of eachdelivery vehicles and determination of their relative abundance.

The pharmaceutical or veterinary composition according to the inventioncan be formulated for any conventional route of administration includinga topical, enteral, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous or intraocular administration and the like,preferably enteral, oral, or inhalation routes.

Then, the pharmaceutical or veterinary composition according to theinvention can be administered by any conventional route ofadministration including a topical, enteral, oral, parenteral,intranasal, intravenous, intramuscular, subcutaneous or intraocularadministration and the like, preferably enteral, oral, intranasal orinhalation routes.

Preferably, the pharmaceutical or veterinary composition according tothe invention may be administered by enteral or parenteral route ofadministration. When administered parenterally, the pharmaceutical orveterinary composition according to the invention is preferablyadministered by intravenous route of administration. When administeredenterally, the pharmaceutical or veterinary composition according to theinvention is preferably administered by oral route of administration.

For oral administration, the pharmaceutical or veterinary compositioncan be formulated into conventional oral dosage forms such as tablets,capsules, powders, granules and liquid preparations such as syrups,elixirs, and concentrated drops. Nontoxic solid carriers or diluents maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, talcum,cellulose, glucose, sucrose, magnesium, carbonate, and the like. Forcompressed tablets, binders, which are agents which impart cohesivequalities to powdered materials, are also necessary. For example,starch, gelatin, sugars such as lactose or dextrose, and natural orsynthetic gums can be used as binders. Disintegrants are also necessaryin the tablets to facilitate break-up of the tablet. Disintegrantsinclude starches, clays, celluloses, algins, gums and crosslinkedpolymers. Moreover, lubricants and glidants are also included in thetablets to prevent adhesion to the tablet material to surfaces in themanufacturing process and to improve the flow characteristics of thepowder material during manufacture. Colloidal silicon dioxide is mostcommonly used as a glidant and compounds such as talc or stearic acidsare most commonly used as lubricants.

For transdermal administration, the pharmaceutical or veterinarycomposition can be formulated into ointment, cream or gel form andappropriate penetrants or detergents could be used to facilitatepermeation, such as dimethyl sulfoxide, dimethyl acetamide anddimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginalsuppositories can be used. The active compounds can be incorporated intoany of the known suppository bases by methods known in the art. Examplesof such bases include cocoa butter, polyethylene glycols (carbowaxes),polyethylene sorbitan monostearate, and mixtures of these with othercompatible materials to modify the melting point or dissolution rate.

Pharmaceutical or veterinary compositions according to the invention maybe formulated to release the active ingredients substantiallyimmediately upon administration or at any predetermined time or timeperiod after administration.

In a particular embodiment, the pharmaceutical or veterinary compositionaccording to the invention further comprises another active ingredient.The additional active ingredient can be a prebiotic, a probiotic, anantibiotic, another antibacterial or antibiofilm agent and/or any agentenhancing the binding of the delivery particle on the bacteria and/orthe delivery of the payload to the bacteria.

Prebiotics include, but are not limited to, amino acids, biotin,fructo-oligosaccharide, galacto-oligosaccharides, hemicelluloses (e.g.,arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin,lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums(e.g., guar gum, gum arabic and carregenaan), oligofructose,oligodextrose, tagatose, resistant maltodextrins (e.g., resistantstarch), trans-galactooligosaccharide, pectins (e.g., xylogalactouronan,citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers(e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber)and xylooligosaccharides.

Probiotics include, but are not limited to lactobacilli, bifidobacteria,streptococci, enterococci, propionibacteria, saccaromycetes,lactobacilli, bifidobacteria, or proteobacteria.

The antibiotic can be selected from the group consisting in penicillinssuch as penicillin G, penicillin K, penicillin N, penicillin O,penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin,cloxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin,hetacillin, bacampicillin, metampicillin, talampicillin, epicillin,carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin;cephalosporins such as cefacetrile, cefadroxil, cephalexin,cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin,cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine,ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam,cefmetazole, cefotetan, cefoxitin, loracarbef, cefbuperazone, cefminox,cefotetan, cefoxitin, cefotiam, cefcapene, cefdaloxime, cefdinir,cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime,cefovecin, cefpimizole, cefpodoxime, cefteram, ceftamere, ceftibuten,ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone,ceftazidime, latamoxef, cefclidine, cefepime, cefluprenam, cefoselis,cefozopran, cefpirome, cefquinome, flomoxef, ceftobiprole, ceftaroline,ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone,cefetrizole, cefivitril, cefmatilen, cefmepidium, cefoxazole, cefrotil,cefsumide, ceftioxide, cefuracetime, and nitrocefin; polymyxins such aspolysporin, neosporin, polymyxin B, and polymyxin E, rifampicins such asrifampicin, rifapentine, and rifaximin; Fidaxomicin; quinolones such ascinoxacin, nalidixic acid, oxolinic acid, piromidic acid, pipemidicacid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin,nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin,balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, temafloxacin,tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin,sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, nemonoxacin,and zabofloxacin; sulfonamides such as sulfafurazole, sulfacetamide,sulfadiazine, sulfadimidine, sulfafurazole, sulfisomidine, sulfadoxine,sulfamethoxazole, sulfamoxole, sulfanitran, sulfadimethoxine,sulfametho-xypyridazine, sulfametoxydiazine, sulfadoxine,sulfametopyrazine, and terephtyl; macrolides such as azithromycin,clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A,josamycin, kitasamycin, midecamycin, oleandomycin, solithromycin,spiramycin, troleandomycin, tylosin, and roxithromycin; ketolides suchas telithromycin, and cethromycin; lluoroketolides such assolithromycin; lincosamides such as lincomycin, clindamycin, andpirlimycin; tetracyclines such as demeclocycline, doxycycline,minocycline, oxytetracycline, and tetracycline; aminoglycosides such asamikacin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin,sisomicin, tobramycin, paromomycin, and streptomycin; ansamycins such asgeldanamycin, herbimycin, and rifaximin; carbacephems such asloracarbef; carbapenems such as ertapenem, doripenem, imipenem (orcilastatin), and meropenem; glycopeptides such as teicoplanin,vancomycin, telavancin, dalbavancin, and oritavancin; lincosamides suchas clindamycin and lincomycin; lipopeptides such as daptomycin;monobactams such as aztreonam; nitrofurans such as furazolidone, andnitrofurantoin; oxazolidinones such as linezolid, posizolid, radezolid,and torezolid; teixobactin, clofazimine, dapsone, capreomycin,cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide,rifabutin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid,metronidazole, mupirocin, platensimycin, quinupristin (or dalfopristin),thiamphenicol, tigecycline, tinidazole, trimethoprim, alatrofloxacin,fidaxomycin, nalidixice acide, rifampin, derivatives and combinationthereof.

The pharmaceutical or veterinary composition according to the inventionmay further comprise a pharmaceutically acceptable vehicle. A solidpharmaceutically acceptable vehicle may include one or more substanceswhich may also act as flavouring agents, lubricants, solubilisers,suspending agents, dyes, fillers, glidants, compression aids, inertbinders, sweeteners, preservatives, dyes, coatings, ortablet-disintegrating agents. Suitable solid vehicles include, forexample calcium phosphate, magnesium stearate, talc, sugars, lactose,dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low meltingwaxes and ion exchange resins.

The pharmaceutical or veterinary composition may be prepared as asterile solid composition that may be suspended at the time ofadministration using sterile water, saline, or other appropriate sterileinjectable medium. The pharmaceutical or veterinary compositions of theinvention may be administered orally in the form of a sterile solutionor suspension containing other solutes or suspending agents (forexample, enough saline or glucose to make the solution isotonic), bilesalts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleateesters of sorbitol and its anhydrides copolymerized with ethylene oxide)and the like. The particles according to the invention can also beadministered orally either in liquid or solid composition form.Compositions suitable for oral administration include solid forms, suchas pills, capsules, granules, tablets, and powders, and liquid forms,such as solutions, syrups, elixirs, and suspensions. Forms useful forenteral administration include sterile solutions, emulsions, andsuspensions.

The bacterial virus particles according to the invention may bedissolved or suspended in a pharmaceutically acceptable liquid vehiclesuch as water, an organic solvent, a mixture of both or pharmaceuticallyacceptable oils or fats. The liquid vehicle can contain other suitablepharmaceutical additives such as solubilisers, emulsifiers, buffers,preservatives, sweeteners, flavouring agents, suspending agents,thickening agents, colours, viscosity regulators, stabilizers orosmo-regulators. Suitable examples of liquid vehicles for oral andenteral administration include water (partially containing additives asabove, e.g. cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols andpolyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.fractionated coconut oil and arachis oil). For parenteraladministration, the vehicle can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid vehicles are useful insterile liquid form compositions for enteral administration. The liquidvehicle for pressurized compositions can be a halogenated hydrocarbon orother pharmaceutically acceptable propellant.

For transdermal administration, the pharmaceutical or veterinarycomposition can be formulated into ointment, cream or gel form andappropriate penetrants or detergents could be used to facilitatepermeation, such as dimethyl sulfoxide, dimethyl acetamide anddimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginalsuppositories can be used. The active compounds can be incorporated intoany of the known suppository bases by methods known in the art. Examplesof such bases include cocoa butter, polyethylene glycols (carbowaxes),polyethylene sorbitan monostearate, and mixtures of these with othercompatible materials to modify the melting point or dissolution rate.

The pharmaceutical or veterinary composition, the bacterial virusparticles as disclosed above and the combination of at least twodifferent bacterial virus particles having the same payload can be usedas a medicament.

The present invention relates to the pharmaceutical or veterinarycomposition, the bacterial virus particles as disclosed above and thecombination of at least two different bacterial virus particles havingthe same payload for use in the treatment of a disorder or a diseasecaused by a bacterium, to the use of the pharmaceutical or veterinarycomposition, the bacterial virus particles as disclosed above and thecombination of at least two different bacterial virus particles havingthe same payload for the manufacture of a medicament useful in thetreatment of a disorder or a disease caused by a bacterium; and to amethod for treating a disorder or a disease caused by a bacteriumcomprising the administration of a therapeutically effective amount ofthe pharmaceutical or veterinary composition, the bacterial virusparticles as disclosed above and the combination of at least twodifferent bacterial virus particles having the same payload. Suchdiseases or disorders include an infection, preferably a bacterialinfection, inflammatory diseases, auto-immune diseases, cancers,metabolic disorders and brain disorders.

In a particular embodiment, the bacterial virus particles only targetthe bacterial strain responsible of the disease or disorder and thusallow the subject to be treated to conserve a healthy microbiome.

The diseases or disorders caused by bacteria may be selected from thegroup consisting of abdominal cramps, acne vulgaris, acute epiglottitis,arthritis, bacteraemia, bloody diarrhea, botulism, Brucellosis, brainabscess, chancroid venereal disease, Chlamydia, Crohn's disease,conjunctivitis, cholecystitis, colorectal cancer, polyposis, dysbiosis,Lyme disease, diarrhea, diphtheria, duodenal ulcers, endocarditis,erysipelothricosis, enteric fever, fever, glomerulonephritis,gastroenteritis, gastric ulcers, Guillain-Barre syndrome tetanus,gonorrhoea, gingivitis, inflammatory bowel diseases, irritable bowelsyndrome, leptospirosis, leprosy, listeriosis, tuberculosis, LadyWidermere syndrome, Legionaire's disease, meningitis, mucopurulentconjunctivitis, multi-drug resistant bacterial infections, multi-drugresistant bacterial carriage, myonecrosis-gas gangrene, Mycobacteriumavium complex, neonatal necrotizing enterocolitis, nocardiosis,nosocomial infection, otitis, periodontitis, phalyngitis, pneumonia,peritonitis, purpuric fever, Rocky Mountain spotted fever, shigellosis,syphilis, sinusitis, sigmoiditis, septicaemia, subcutaneous abscesses,tularaemia, tracheobronchitis, tonsillitis, typhoid fever, ulcerativecolitis, urinary infection, whooping cough.

The infection caused by bacteria may be selected from the groupconsisting of skin infections such as acne, intestinal infections suchas esophagitis, gastritis, enteritis, colitis, sigmoiditis, rectitis,and peritonitis, urinary tract infections, vaginal infections, femaleupper genital tract infections such as salpingitis, endometritis,oophoritis, myometritis, parametritis and infection in the pelvicperitoneum, respiratory tract infections such as pneumonia,intra-amniotic infections, odontogenic infections, endodonticinfections, fibrosis, meningitis, bloodstream infections, nosocomialinfection such as catheter-related infections, hospital acquiredpneumonia, post-partum infection, hospital acquired gastroenteritis,hospital acquired urinary tract infections, or a combination thereof.Preferably, the infection according to the invention is caused by abacterium presenting an antibiotic resistance. In a particularembodiment, the infection is caused by a bacterium as listed above inthe targeted bacteria.

The metabolic disorder includes obesity and diabetes.

In a particular embodiment, the invention concerns a pharmaceutical orveterinary composition for use in the treatment of pathologies involvingbacteria of the human microbiome, such as inflammatory and auto-immunediseases, cancers, infections or brain disorders. Indeed, some bacteriaof the microbiome, without triggering any infection, can secretemolecules that will induce and/or enhance inflammatory or auto-immunediseases or cancer development. More specifically, the present inventionrelates also to modulating microbiome composition to improve theefficacy of immunotherapies based, for example, on CAR-T (ChimericAntigen Receptor T) cells, TIL (Tumor Infiltrating Lymphocytes) andTregs (Regulatory T cells) also known as suppressor T cells. Modulationof the microbiome composition to improve the efficacy of immunotherapiesmay also include the use of immune checkpoint inhibitors well known inthe art such as, without limitation, PD-1 (programmed cell deathprotein 1) inhibitor, PD-L1 (programmed death ligand 1) inhibitor andCTLA-4 (cytotoxic T lymphocyte associated protein 4).

Some bacteria of the microbiome can also secrete molecules that willaffect the brain.

Therefore, a further object of the invention is a method for controllingthe microbiome of a subject, comprising administering an effectiveamount of the pharmaceutical composition as disclosed herein in saidsubject.

In a particular embodiment, the invention also relates to a method forpersonalized treatment for an individual in need of treatment for abacterial infection comprising: i) obtaining a biological sample fromthe individual and determining a group of bacterial DNA sequences fromthe sample; ii) based on the determining of the sequences, identifyingone or more pathogenic bacterial strains or species that were in thesample; and iii) administering to the individual a pharmaceuticalcomposition according to the invention comprising a combination of atleast two bacterial virus particles capable of recognizing eachpathogenic bacterial strain or species identified in the sample and todeliver the packaged payload.

Preferably, the biological sample comprises pathological andnon-pathological bacterial species, and subsequent to administering thepharmaceutical or veterinary composition according to the invention tothe individual, the amount of pathogenic bacteria on or in theindividual are reduced, but the amount of non-pathogenic bacteria is notreduced.

In another particular embodiment, the invention concerns apharmaceutical or veterinary composition according to the invention foruse in order to improve the effectiveness of drugs. Indeed, somebacteria of the microbiome, without being pathogenic by themselves, areknown to be able to metabolize drugs and to modify them in ineffectiveor harmful molecules.

In another particular embodiment, the invention concerns the in-situbacterial production of any compound of interest, including therapeuticcompound such as prophylactic and therapeutic vaccine for mammals. Thecompound of interest can be produced inside the targeted bacteria,secreted from the targeted bacteria or expressed on the surface of thetargeted bacteria. In a more particular embodiment, an antigen isexpressed on the surface of the targeted bacteria for prophylacticand/or therapeutic vaccination.

The present invention also relates to a non-therapeutic use of thebacterial virus particles as disclosed above and the combination of atleast two different bacterial virus particles having the same payloadaccording to the invention. For instance, the non-therapeutic use can bea cosmetic use or a use for improving the well-being of a subject, inparticular a subject who does not suffer from a disease. Accordingly,the present invention also relates to a cosmetic composition or anon-therapeutic composition comprising the bacterial virus particles asdisclosed above and the combination of at least two different bacterialvirus particles having the same payload according to the invention.

Subject, Regimen and Administration

The subject according to the invention is an animal, preferably amammal, even more preferably a human. However, the term “subject” canalso refer to non-human animals, in particular mammals such as dogs,cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, gerbils,hamsters, chinchillas, rats, mice, guinea pigs and non-human primates,among others, or non-mammals such as poultry, that are in need oftreatment.

The human subject according to the invention may be a human at theprenatal stage, a new-born, a child, an infant, an adolescent or anadult at any age.

In a preferred embodiment, the subject has been diagnosed with, or is atrisk of developing an infection, a disorder and/or a disease preferablydue to a bacterium. Diagnostic method of such infection, disorder and/ordisease are well known by the man skilled in the art.

In a particular embodiment, the infection, disorder and/or diseasepresents a resistance to treatment, preferably the infection, disorderor disease presents an antibiotic resistance.

In a particular embodiment, the subject has never received any treatmentprior to the administration of the delivery vehicles according to theinvention, preferably a payload according to the invention, particularlya payload packaged into a delivery vehicle according to the invention,preferably a packaged plasmid or phagemid into a bacterial virusparticle according to the invention, or of a pharmaceutical orveterinary composition according to the invention.

In a particular embodiment, the subject has already received at leastone line of treatment, preferably several lines of treatment, prior tothe administration of the delivery vehicles according to the invention,preferably a payload according to the invention, particularly a payloadpackaged into a delivery vehicle according to the invention, preferablya packaged plasmid or phagemid into a bacterial virus particle accordingto the invention, or of a pharmaceutical or veterinary compositionaccording to the invention.

Preferably, the treatment is administered regularly, preferably betweenevery day and every month, more preferably between every day and everytwo weeks, more preferably between every day and every week, even morepreferably the treatment is administered every day. In a particularembodiment, the treatment is administered several times a day,preferably 2 or 3 times a day, even more preferably 3 times a day.

The duration of treatment with delivery vehicles according to theinvention, preferably a payload according to the invention, particularlya payload packaged into a delivery vehicle according to the invention,preferably a packaged plasmid or phagemid into a bacterial virusparticle according to the invention, or with a pharmaceutical orveterinary composition according to the invention, is preferablycomprised between 1 day and 20 weeks, more preferably between 1 day and10 weeks, still more preferably between 1 day and 4 weeks, even morepreferably between 1 day and 2 weeks. In a particular embodiment, theduration of the treatment is of about 1 week. Alternatively, thetreatment may last as long as the infection, disorder and/or diseasepersists.

The form of the pharmaceutical or veterinary compositions, the route ofadministration and the dose of administration of delivery vehiclesaccording to the invention, preferably of a payload according to theinvention, particularly of a payload packaged into a delivery vehicleaccording to the invention, preferably of a packaged plasmid or phagemidinto a bacterial virus particle according to the invention, or of apharmaceutical or veterinary composition according to the invention canbe adjusted by the man skilled in the art according to the type andseverity of the infection (e.g. depending on the bacteria speciesinvolved in the disease, disorder and/or infection and its localizationin the patient's or subject's body), and to the patient or subject, inparticular its age, weight, sex, and general physical condition.

Particularly, the amount of delivery vehicles according to theinvention, preferably a payload according to the invention, particularlya payload packaged into a delivery vehicle according to the invention,preferably a packaged plasmid or phagemid into a bacterial virusparticle according to the invention, or of a pharmaceutical orveterinary composition according to the invention, to be administeredhas to be determined by standard procedure well known by those ofordinary skills in the art. Physiological data of the patient or subject(e.g. age, size, and weight) and the routes of administration have to betaken into account to determine the appropriate dosage, so as atherapeutically effective amount will be administered to the patient orsubject.

For example, the total amount of delivery vehicles, particularly apayload packaged into a delivery vehicle according to the invention,preferably a plasmid or phagemid packaged into a bacterial virusparticle according to the invention, for each administration iscomprised between 10⁴ and 10¹⁵ delivery vehicles.

EXAMPLES—RESULTS

The inventors have shown single payload encapsidation in three differentcapsids from three different phage families: P1-like (P1), P2-like (186)and lambdoid (lambda). P1 relies on a headfull packaging system whereasboth P2-like and Lambdoid rely on cohesive end packaging. Even if theyuse the same phage termini type (cohesive ends) lambdoid and P2-like usea different DNA substrate for the packaging. Indeed, lambdoid phagesundergo rolling circle replication to generate a concatemer of the phagegenome (head to tail repeat of the DNA), then the terminase bind on thepackaging recognition site called cos, generate a double strandcleavage, get recruited on the procapsid and start to fulfill it in aunidirectional process until the next cos. For P2, the preferred DNAsubstrate is a circular monomer, which is recognized by the terminase atthe cos level and then cleaved and packaged. Consequently, a monomericcircular DNA molecule that is suitable for P2 packaging and can also getconcatemerized for lambda packaging was used. For phages relying onheadful packaging such as P1, chromosomes are circularly permuted andcontain terminally redundancy that allow recircularization of DNA uponinjection. Then the packaging site called pac is recognized by theterminase (or pacase) that attaches to a procapsid and fulfill more than100% of the genome and cut.

Construction and Test

To test the packaging of a single payload into 3 different phagecapsids, a lambda cos site, a cos site recognized by 186 terminase and aP1 pac site were cloned into a single plasmid and transformed into 3different E. coli production strains:

-   -   CY2120b (Cronan et al, 2013, Plasmid 69: 81-89) a lysogen of        lambda lacking the wild-type lambda cos site    -   C600(186)Δcos a C600 lysogen of 186 lacking the wild-type 186        cos site    -   KL739 a C600 lysogen of P1

Along with the 3 different packaging sites, the plasmid includes achloramphenicol resistance gene that allows scoring for transductant, acolE1 origin of replication, the Coi gene under the control of pBad thatallows induction of the lytic cycle of P1, a cis element inside the cingene of P1 and the P1 replication origin inside repL.

The three different lysogene cell lines containing the payload wereinduced to produce phagemids particles. For the production cell lineCY2120b and C600(186)Δcos only phagemids particles and no phages wereproduced due to the deletion of the cos site from the prophages. For thestrain KL739, a mix of a phage and phagemid particles were produced.

Titrations of the pure phagemids particles were done on strain E. coliK-12 MG1655 whereas titration of the phagemid particles mixed withphages where done on the lysogene itself (KL739) allowing titration ofphagemids particles without killing the bacteria. Count of transductanton chloramphenicol was performed the day after.

The entire test, i.e. induction and titration, was performed intriplicate. The graphs of FIG. 1 represent the mean of the phagemidparticle concentration per μL obtained for each phagemids particles in 3independent experiments as detailed in FIG. 2.

The results show that a single plasmid is able to get packaged and givesviable phagemids particles for all the three different capsids. Thenumber of particles varies from 2.53 10⁷ for lambda, 7.77 10⁵ for 186 to7.9 10⁴ for P1. Such differences can be explained by the competitionbetween the P1 phage still containing the pac site and the plasmid forthe packaging.

In conclusion, it has been demonstrated that adding several packagingsignals (pac, cos) from phages having different packaging mechanismsallows for the production of phagemids particles with the same DNAplasmid but different capsids.

MATERIAL AND METHODS

Production of Phagemids Particles

The three different cell lines containing the payload were grownseparately overnight in LB+chloramphenicol 25 μg/mL. The following day,cells were diluted 1/100 in 10 mL of LB and incubated at 30° C. withshaking. For strain CY2120b and C600(186)Δcos the culture, at OD600 nmaround 0.6, were shifted to 42° C. for 30 minutes to induce the entryinto lytic cycle. After that, cells were shifted back to 37° C. for 2hours (C600(186)Δcos) or 3 hours (CY2120b) to allow virion assembly andpackaging in either of the two capsids. Chlorophorm was added in thecase of CY2120b to burst the cell. Cell lysate was filtered with 0.2 μmsyringe filter.

For the KL739 production, the induction of the lytic cycle was performedby adding arabinose 0.2%. After 2 hours cell lysate was filtered with0.2 μm syringe filter.

Titration of Phagemids Particles

E. coli KL739 and E. coli K-12 MG1655 overnight culture in LB wasdiluted 1/100. At OD600 nm between 0.6 and 0.8, 90 μl of cells wereincubated at 37° C. for 30 min with 10 μL of each phagemids diluted1/100. After incubation serial dilution of the samples were performed inPBS 1× and all dilution were plated on LB supplemented withchloramphenicol 25 μg/mL. Plate were incubated at 37° C. overnight andthe CFU counts done the day after.

REFERENCES

-   Abudayyeh, O., et al. (2017). RNA targeting with CRISPR-Cas13.    Nature 550, 280.-   Cronan, J. Improved plasmid-based system for fully regulated    off-to-on gene expression in Escherichia coli: Application to    production of toxic proteins. Plasmid 69, 81-89 (2013).-   Fonfara, I., et al. (2014). Phylogeny of Cas9 determines functional    exchangeability of dual-RNA and Cas9 among orthologous type II    CRISPR-Cas systems. Nucleic Acids Research 42, 2577-2590.-   Koonin, E., et al. (2017). Diversity, classification and evolution    of CRISPR-Cas systems. Curr Opin Microbiol 37, 67-78.-   Krom, R., Bhargava, P., Lobritz, M. & Collins, J. Engineered    Phagemids for Nonlytic, Targeted Antibacterial Therapies. Nano    Letters 15, 4808-4813 (2015).-   Yan, W., et al. (2018). Cas13d Is a Compact RNA-Targeting Type VI    CRISPR Effector Positively Modulated by a WYL-Domain-Containing    Accessory Protein. Mol Cell.-   Krupovic, M., et al. (2015). Taxonomy of prokaryotic viruses: update    from the ICTV bacterial and archaeal viruses subcommittee. Arch    Virol 161, 1095-1099.-   B Hohn and K Murray (1977). Packaging recombinant DNA molecules into    bacteriophage particles in vitro. PNAS 74(8): 3259-3263.-   John Collins and Barbara Hohn (1978). Cosmids: A type of plasmid    gene-cloning vector that is packageable in vitro in bacteriophage λ    heads. PNAS 75(9): 4242-4246.-   E J Gunther, et al. (1993). High efficiency, restriction-deficient    in vitro packaging extracts for bacteriophage lambda DNA using a    new E. coli lysogen. NAR 21(16): 3903-3904.

What is claimed is:
 1. A pharmaceutical composition comprising at leasttwo different bacterial delivery vehicles into which the same payload ispackaged, wherein the payload comprises: a nucleic acid sequence ofinterest encoding a therapeutic molecule of interest, under the controlof a promoter; and at least two orthogonal bacterial virus packagingsites that allow packaging of said payload into said at least twodifferent bacterial delivery vehicles and a pharmaceutical acceptablecarrier, wherein the nucleic acid sequence of interest encodes one ormore components of a Cas9 system for the reduction of gene expression orinactivation of a gene, wherein said gene is selected from the groupconsisting of an antibiotic resistance gene, a virulence factor orvirulence protein gene, a toxin factor or toxin protein gene, abacterial receptor gene, a membrane protein gene, a structural proteingene, a secreted protein gene, and a drug resistance gene, or anycombination thereof.
 2. The pharmaceutical composition according toclaim 1, wherein the at least two orthogonal bacterial virus packagingsites are selected from the group consisting of: (i) at least twodifferent cos sites; (ii) at least two different pac sites; (iii) atleast two different concatemer junction sites; (iv) at least one cossite and at least one pac site; (v) at least one cos site and at leastone concatemer junction site; (vi) at least one pac site and at leastone concatemer junction site; and (vii) at least one cos site, at leastone pac site and at least one concatemer junction site.
 3. Thepharmaceutical composition according to claim 1, wherein the at leasttwo orthogonal bacterial virus packaging sites are selected from thegroup consisting of: a λ cos site, a P4 cos site, a SPP1 pac site, a P1pac site, a T1 pac site, a mu pac site, a P22 pac site, a φ8 pac site, aSf6 pac site, a 149 pac site, a T7 concatemer junction, and an μl122-concatemer junction.
 4. The pharmaceutical composition according toclaim 3, wherein the at least two orthogonal bacterial virus packagingsites comprise: (i) λ cos site and P4 cos site; (ii) a λ cos site, P4cos site and P1 pac site; (iii) a λ cos site, P4 cos site and T7concatemer junction; or (iv) a λ cos site, P4 cos site, P1 pac site andT7 concatemer junction.
 5. The pharmaceutical composition according toclaim 1, wherein the bacterial delivery vehicles are bacterial viruses.6. The pharmaceutical composition according to claim 5, wherein thebacterial viruses are selected from the group consisting of BW73, B278,D6, D108, E, E1, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu,025, PhI-5, Pk, PSP3, P1, P1D, P2, P4, S1, Wφ, φK13, φ1, φ2, φ7, φ92, 7A, 8φ, 9φ, 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM, C4, C16,DD-VI, E4, E7, E28, FI1, FI3, H, H1, H3, H8, K3, M, N, ND-2, ND-3, ND4,ND-5, ND6, ND-7, Ox-I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, PhI-I, RB42, RB43,RB49, RB69, S, SaI-I, Sal-2, Sal-3, Sal-4, Sal-5, Sal-6, TC23, TC45,TuII*-6, TuIP-24, TuII*46, TuIP-60, T2, T4, T6, T35, αl, 1, IA, 3, 3A,3T+, 5φ, 9266Q, CFO103, HK620, J, K, K1F, m59, no. A, no. E, no. 3, no.9, N4, sd, T3, T7, WPK, W31, AH, φC3888, φK3, φK7, φK12, φV-1, Φ04-CF,Φ05, Φ06, Φ07, φ1, φ1.2, φ20, φ95, φ263, φ1O92, φ1, φ11, Ω28, 1, 3, 7,8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W, NN-Escherichia (1),Esc-7-11, AC30, CVX-5, Cl, DDUP, EC1, EC2, E21, E29, F1, F26S, F27S, Hi,HK022, HK97, HK139, HK253, HK256, K7, ND-I, PA-2, q, S2, T1, T3C, T5,UC-I, w, β4, γ2, λ, ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ, 2, 4, 4A, 6, 8A,102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95,HK243, K1O, ZG/3A, 5, 5A, 21EL, H19-J and 933H.
 7. The pharmaceuticalcomposition according to claim 1, wherein the bacterial deliveryvehicles are capable of targeting at least two different bacteria and ofintroducing the payload into the at least two different bacteria.
 8. Thepharmaceutical composition according to claim 1, wherein the bacterialdelivery vehicles are capable of targeting the same bacteria and ofintroducing the payload into the same bacteria.
 9. The pharmaceuticalcomposition according to claim 1, wherein the pharmaceutical compositionfurther comprises at least one additional active ingredient.
 10. Thepharmaceutical composition according to claim 9 wherein the additionalactive ingredient is selected from the group consisting of a prebiotic,a probiotic, an antibiotic, an antibacterial agent, an antibiofilmagent, an agent that enhances the targeting of the bacterial deliveryvehicles to a bacteria and, an agent that enhances the delivery of thepayload into the bacteria, or any combination thereof.
 11. Apharmaceutical composition comprising at least two different bacterialdelivery vehicles into which the same payload is packaged, wherein thepayload comprises: a nucleic acid sequence of interest encoding atherapeutic molecule of interest, under the control of a promoter; andat least two orthogonal bacterial virus packaging sites that allowpackaging of said payload into said at least two different bacterialdelivery vehicles and a pharmaceutical acceptable carrier, wherein theat least two orthogonal bacterial virus packaging sites comprise: (i) aλ cos site and P4 cos site; (ii) a λ cos site, P4 cos site and P1 pacsite; (iii) a λ cos site, P4 cos site and T7 concatemer junction; or(iv) a λ cos site, P4 cos site, P1 pac site and T7 concatemer junction;and wherein the nucleic acid sequence of interest is selected from thegroup consisting of a nucleic acid sequence encoding a kinase, anuclease, a Cas nuclease, a Cas9 nuclease, a guide RNA, a CRISPR locus,a toxin, a TALEN, a ZFN, a meganuclease, a recombinase, a bacterialreceptor, a membrane protein, a structural protein, a secreted protein,a gene product conferring resistance to an antibiotic or to a drug, atoxic protein or a toxic factor, a virulence protein and a virulencefactor, or any combination thereof.
 12. The pharmaceutical compositionaccording to claim 11, wherein the bacterial delivery vehicles arebacterial viruses.
 13. The pharmaceutical composition according to claim12, wherein the bacterial viruses are selected from the group consistingof BW73, B278, D6, D108, E, E1, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B,i, MM, Mu, 025, PhI-5, Pk, PSP3, P1, P1D, P2, P4, S1, Wφ, φK13, φ1, φ2,φ7, φ92, 7 A, 8φ, 9φ, 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM,C4, C16, DD-VI, E4, E7, E28, FI1, FI3, H, H1, H3, H8, K3, M, N, ND-2,ND-3, ND4, ND-5, ND6, ND-7, Ox-I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, PhI-I,RB42, RB43, RB49, RB69, S, SaI-I, Sal-2, Sal-3, Sal-4, Sal-5, Sal-6,TC23, TC45, TuII*-6, TuIP-24, TuII*46, TuIP-60, T2, T4, T6, T35, α1, 1,IA, 3, 3A, 3T+, 5φ, 9266Q, CFO103, HK620, J, K, K1F, m59, no. A, no. E,no. 3, no. 9, N4, sd, T3, T7, WPK, W31, ΔH, φC3888, φK3, φK7, φK12,φV-1, Φ04-CF, Φ05, Φ06, Φ07, φ1, φ1.2, φ20, φ95, φ263, φ1O92, φ1, φ11,Ω8, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W,NN-Escherichia (1), Esc-7-11, AC30, CVX-5, C1, DDUP, EC1, EC2, E21, E29,F1, F26S, F27S, Hi, HK022, HK97, HK139, HK253, HK256, K7, ND-I, PA-2, q,S2, T1, T3C, T5, UC-I, w, (β4, γ2, λ, ΦD326, φγ, Φ06, Φ7, Φ10, Φ80, χ,2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50,AC57, AC81, AC95, HK243, K1O, ZG/3A, 5, 5A, 21EL, H19-J and 933H. 14.The pharmaceutical composition according to claim 11, wherein thebacterial delivery vehicles are capable of targeting at least twodifferent bacteria and of introducing the payload into the at least twodifferent bacteria.
 15. The pharmaceutical composition according toclaim 11, wherein the bacterial delivery vehicles are capable oftargeting the same bacteria and of introducing the payload into the samebacteria.
 16. The pharmaceutical composition according to claim 11,wherein the pharmaceutical composition further comprises at least oneadditional active ingredient.
 17. The pharmaceutical compositionaccording to claim 16 wherein the additional active ingredient isselected from the group consisting of a prebiotic, a probiotic, anantibiotic, an antibacterial agent, an antibiofilm agent, an agent thatenhances the targeting of the bacterial delivery vehicles to a bacteriaand, an agent that enhances the delivery of the payload into thebacteria, or any combination thereof.